Vesicular monoamine transporter gene therapy in Parkinson&#39;s disease

ABSTRACT

The present invention provides methods and compositions for the therapeutic intervention of Parkinson&#39;s disease. More particularly, methods of making and sequestering dopamine are disclosed. Additionally, methods of genetically modifying donor cells by gene transfer for grafting into the central nervous system to treat defective, diseased or damaged cells are disclosed. Methods and compositions for carrying out such gene transfer and grafting are described.

The present application claims the benefit of U.S. Provisional PatentApplication, Serial No. 60/112,502, filed Dec. 16, 1998. The governmentowns rights in the present invention pursuant to grant number NS32080from the National Institutes of Health.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of neurobiologyand biochemistry. More particularly, it concerns methods andcompositions for therapeutic intervention against Parkinson's disease.In particular, methods of making and sequestering dopamine aredisclosed.

2. Description of Related Art

Parkinson's disease (PD) is an age-related disorder characterized by aloss of dopamine neurons in the substantia nigra of the midbrain. Theseneurons have the basal ganglia as their major target organ. The symptomsof PD include tremor, rigidity and ataxia. The disease is progressive,but can be treated by replacement of dopamine through the administrationof pharmacological doses of the precursor for dopamine,L-3,4-dihydroxyphenylalanine (levodopa; L-DOPA; Marsden, 1986; Vinken etal., 1986). However, with chronic use of this pharmacotherapy, thepatients often develop a fluctuating response to L-DOPA. There are manysuggested mechanisms for the development of the fluctuation, but onesimple explanation is that the patients reach a threshold of cell andterminal loss, wherein the remaining cells cannot synthesize and storesufficient dopamine from the precursor.

Typically PD patients are routinely treated with a combination of L-DOPAand a DOPA decarboxylase inhibitor such as carbidopa or benserazide.Unfortunately, after an initial period of satisfactory, smooth andstable clinical benefit from L-DOPA therapy, lasting on the average 2-5years, the condition of many patients deteriorates and they developcomplex dose-related, as well as unpredictable, response fluctuations.The causes of the response fluctuations are probably multiple andcomplex, but pharmacokinetic problems likely play some role. There is acorrelation between the clinical fluctuations and the oscillations ofL-DOPA plasma levels. However, many of the problems are a result of theunfavorable pharmacodynamics response to L-DOPA, i.e., a short half-lifein vivo due to various central factors.

Another treatment route is through intracerebral grafting. PD is thefirst disease of the brain for which therapeutic intracerebral graftinghas been used in humans. Preclinical and clinical data indicate thattransplanted cells (the graft) used in cell transplantation protocolsfor these types of neurodegenerative diseases survive and integrate withthe host tissue, and provides functional recovery (Wictorin et al.,1990).

Several attempts have been made to provide the neurotransmitter dopamineto cells of the basal ganglia of Parkinson's patients by transplantationof fetal brain cells from the substantia nigra, an area of the brainrich in dopamine-containing cell bodies and also the area of the brainmost affected in PD. Fetal dopaminergic neurons have been shown to beeffective in reversing the behavioral deficits in rat models of PDinduced by selective dopaminergic neurotoxins (Bjorklund et al., 1986;Dunnett et al., 1983). The major effect of fetal transplantation in PDhas been in enhancing patients' response to L-DOPA, rather thanalleviating the need for the drug. This effect is thought to be due toadded capacity to decarboxylate L-DOPA and storage of the formeddopamine (Lindvall et al, 1994; Sawle et al, 1992).

Non-fetal cell transplants also have been used in an attempt to combatPD, e.g., the use of chromaffin cells. A major advantage of this type oftransplantation protocol is that the graft source is not a fetal sourceand thus, circumvents the ethical and logistical problems associatedwith acquiring fetal tissue. Using the chromaffin cell protocol,normalization of behavior has been observed. However, the functionalrecovery of this behavior is temporary and the animals revert to theirpre-transplantation status (Bjorklund and Stenevi, 1985; Lindvall etal., 1987). The inability of this type of treatment protocol to maintainnormal behavioral activity in animals in the PD model renders clinicalapplication of this protocol as well as other treatment therapiesineffective.

Finally, it is known that the chemical deficit that results in PD is theinability to supply dopamine. The rate limiting enzyme in the productionof dopamine, tyrosine hydroxylase, has been cloned and the anatomicallocalization of the affected region has been identified as the basalganglia. Neverthless, in animal models, it has been shown thatincreasing the activity of tyrosine hydroxylase in Parkinsonian tissuedoes not result in the long-term amelioration of the symptoms of PD.

Clearly, there is a need for methods and compositions that will correctthe chemical deficit in PD in a reliable manner that will lead to thetreatment of the disease.

SUMMARY OF THE INVENTION

The present invention provides a method for producing dopamine in a cellcomprising transforming a cell with a first polynucleotide encodingaromatic L-amino acid decarboxylase (AADC) and a second polynucleotideencoding vesicular monoamine transporter (VMAT) under conditionssuitable for the expression of AADC and VMAT, wherein thepolynucleotides each are under the transcriptional control of a promoteractive in eukaryotic cells; and contacting the cell withL-3,4-dihydroxyphenylalanine (L-DOPA), whereby AADC converts L-DOPA todopamine and VMAT sequesters the dopamine in endosomes in the cell. Incertain preferred embodiments, the first and second polynucleotides arecovalently attached. It is contemplated that the first and secondpolynucleotides are part of a viral vector. In alternative embodiments,first and second polynucleotides are not covalently attached. It may bethat the first and second polynucleotides are part of first and secondviral vectors, respectively. It is contemplated that the viral vectormay be selected from the group consisting of retrovirus, adenovirus,herpes virus, adeno-associated virus and lentivirus.

In those embodiments in which the first and second polynucleotides arecovalently attached it is contemplated that the first and secondpolynucleotides may be under control of different promoters or under thecontrol of the same promoter. In those embodiments in which the firstand second polynucleotides are under the control of the same promoter,the first and second polynucleotides may be separated by an internalribosome entry site.

It is contemplated that as used herein the promoter may be a tissuespecific promoter, an inducible promoter or a constitutive promoter.Such promoters are described throughout the application and are wellknown to those of skill in the art. In particularly preferredembodiments, the promoter may be selected from the group consisting ofCMV IE, SV40 IE, β-actin, EF1-α, a TH promoter, AADC non-neuronalpromoter; an AADC promoter, a VMAT2 promoter region, a GTPcyclohydrolase I promoter and a dopamine transporter promoter. Of coursethese and the other promoters listed throughout the specification areexemplary promoters and one of skill in the art may well substituteother promoter regions for those listed herein and still achieve theobjectives of the present invention.

In those embodiments in which the first and second polynucleotides arecovalently linked, it is contemplated that the first and secondpolynucleotides each are covalently linked to a polyadenylation signal.

In particularly preferred embodiments, the cell is a fibroblast cell. Inother embodiments the cell may be a fetal brain cell, astrocytes,neuronal stem cells, neuronal precursor cells, myoblasts, bone marrowstromal cells.

In certain aspects of the present invention, it is contemplated that themethod further may comprise transforming the cell with a polynucleotideencoding tyrosine hydroxylase (TH) wherein the TH encodingpolynucleotide is under the transcriptional control of a promoter. Instill further embodiments, the method further may comprise transformingthe cell with a polynucleotide encoding GTP cyclohydrolase I (GTPCH) inaddition to the polynucleotide encoding TH, wherein the GTPCHpolynucleotide is are under the transcriptional control of a promoter.

In specific embodiments, the cell may be transformed in vivo. In otherembodiments, the cell is transformed ex vivo and implanted into asubject. In those embodiments in which the cell is implanted into asubject, the L-DOPA may be administered to the patient using any routecommonly recommended by a physician. More particularly, it iscontemplated that the L-DOPA may be administered orally, subcutaneouslyor sublingually. In certain defined embodiments, the cell is derivedfrom the subject prior to transformation.

Another aspect of the present invention contemplates a method oftreating Parkinson's disease in a subject comprising obtaining a cellsfrom the subject; transforming the cells with a first polynucleotideencoding L-amino acid decarboxylase (AADC) and a second polynucleotideencoding VMAT under conditions suitable for the expression of AADC andVMAT, wherein the polynucleotides each are under the transcriptionalcontrol of a promoter; implanting transformed cells into the subject;and providing L-DOPA to the subject, whereby AADC converts L-DOPA invivo to dopamine and VMAT sequesters the dopamine in endosomes in thecell, which sequestered dopamine over a longer duration of time thanfrom cells without storage of L-DOPA. In particularly preferredembodiments, the L-DOPA is administered orally, sublingually,subcutaneously, intravenously or by duodenal infusion.

It is contemplated that the dose of L-DOPA to be administered may fallwithin the range of any L-DOPA therapy currently in use or anyconcentration that becomes useful in future. It is particularlycontemplated that the L-DOPA is administered in a dose of between about50 mg to about 2500 mg of L-DOPA per day. Thus it is contemplated that50 mg, 75 mg, 100 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg,550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg,1000 mg, 1100 mg, 1200 mg, 1250 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg,1700 mg, 1800 mg, 1900 mg, 2000 mg, 2100 mg, 2200 mg, 2300 mg, 2400 mg,2500 mg of L-DOPA per day or more. This dose of L-DOPA may beadministered in one dose or alternatively may be split into several (2,3, 4, 5 or more) aliquots to be administered over the period of 24hours. Alternatively, it also is contemplated that the L-DOPA may beadministered continuously throughout the day. It is contemplated thatthe L-DOPA is administered alone or alternatively may be administered incombination with carbidopa and/or any other PD therapeutic agent asdescribed in the specification below. In particularly preferredembodiments, the L-DOPA is administered in combination with carbidopa ata dose of between about 20 mg to about 300 mg carbidopa per day. Thus itis contemplated that 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180mg 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270mg, 280 mg, 290 mg, 300 mg carbidopa per day or more may beadministered. This may be administered in a single dose or in multipledoses throughout the day.

In specific embodiments, it is contemplated that the transformed cellsare implanted via stereostaxic surgery.

Also contemplated by the present invention is a kit comprising apolynucleotide encoding aromatic L-amino acid decarboxylase (AADC); anda polynucleotide encoding vesicular monoamine transporter (VMAT), thepolynucleotides being located in suitable container means therefor.

In preferred embodiments, the VMAT is VMAT type II. In other preferredembodiments, the VMAT is VMAT type I. In specifically definedembodiments, the polynucleotides each are part of a viral vector andeach are under the transcriptional control of a promoter active ineukaryotic cells. In particular embodiments, the viral vector isselected from the group consisting of retrovirus, adenovirus, herpesvirus, adeno-associated virus and lentivirus. In certain preferredembodiments the kit comprises L-DOPA in a suitable container meanstherefor.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1A-FIG. 1F. Transgene expression in genetically modifiedfibroblasts. The top row (FIG. 1A to FIG. 1C) shows examples of AADCimmunohistochemistry. Both PFAADC (FIG. 1B) and PFVMAA (FIG. 1C) showimmunoreactivity to AADC antibody. The bottom row (FIG. 1D to FIG. 1F)shows example of VMAT-2 immunohistochemistry. Only PFVMAA (FIG. 1F)shows immunoreactivity to VMAT-2 especially in perinuclear area. PF(FIG. 1A and FIG. 1D) does not show any immunoreactivity to AADC orVMAT-2 antibody. The scale bar equals 500 μm.

FIG. 2A-FIG. 2D. Dopamine production and storage in genetically modifiedfibroblast cells from exogenous L-DOPA. Time course of intracellular(FIG. 2A) and extracellular (FIG. 2B) dopamine levels in PFAADC cells(open circle) and PFVMAA cells (solid circle) after 1 μM L-DOPAincubation for various duration (0 to 6 hours). The dopamine (FIG. 2C)and DOPAC (FIG. 2D) levels in PFAADC cells and PFVMAA cells after 2hour-incubation with 1 μM L-DOPA and 3 μM reserpine (+rsp) or withoutreserpine (−rsp). The catecholamine levels were measured both in themedia (solid bar) and in the cells (open bar). Data represent themean±SEM (n=3), from a representative set of several experiments. n.d.,nondetectable levels. * p<0.01, Newman-Keuls post-hoc analysis.

FIG. 3A and FIG. 3B. Release of stored dopamine from PFVMAA. (FIG. 3A)Time course of dopamine release from PFVMAA cells that were preincubatedwith 1 μM of L-DOPA for 2 hours to store dopamine (intracellular: openbar). After pre-incubation, the media was replaced with fresh mediawithout L-DOPA and dopamine levels in the media (solid bar) weremeasured at one hour intervals. (FIG. 3B) The dopamine levels in themedia (solid bar) and in the cell pellets (open bars) of PFVMAA cells indifferent calcium conditions. Cells were preincubated with 1 μM L-DOPAin the media for 3 hours, which were replaced with fresh physiologicalmedia (control), physiological media with 20 μM A23187 (Sigma), or bycalcium-free media with 10 mM EGTA for 20 minutes of further incubation.Data represent the mean±SEM (n=3). n.d., nondetectable levels. n.s., nosignificant, * p<0.01, Student t-test analysis.

FIG. 4A-FIG. 4D. Time course of biochemical and behavioral changes afterL-DOPA injection. Microdialysate concentrations of L-DOPA (FIG. 4A),dopamine (FIG. 4B), DOPAC (FIG. 4C) were measured in 6-OHDA-denervatedstriatum containing genetically modified grafts at 20 min intervalsafter L-DOPA i.p. injection (6 mg/kg except for one PFAADC group whichreceived 25 mg/kg). Data represent the mean±SEM (n=3 for no graftcontrol, n=6 for PF, n=4 for PFAADC with 6 mg/kg, n=5 for PFAADC with 25mg/kg, n=6 for PFVMAA). †p<0.05, * p<0.01 relative to all other groupsin FIG. 4A, relative to AADC 25 mg/kg group in FIG. 4B, relative to PF,no graft, and PFAADC groups given 6 mg/kg of L-DOPA in FIG. 4C, byNewman-Keuls post-hoc analysis. (FIG. 4D) Time course of thecontralateral forepaw adjusting steps after L-DOPA administration (6mg/kg). Data represent the mean±SEM (n=6 for PF, n=8 for PFAADC, n=11for PFVMAA). †p<0.05, * p<0.01 relative to pre-L-DOPA step numbers byNewman-Keuls post-hoc analysis. Benserazide was given at 25 mg/kg i.p.in all groups for both biochemical and behavioral measures.

FIG. 5A-FIG. 5H. Immunohistochemical staining of genetically modifiedgrafts in dopamine-denervated striatum (24). The top row (FIG. 5A toFIG. 5C) shows examples of AADC immunohistochemistry. In the bottom row,FIG. 5E and FIG. 5F show example of VMAT-2 immunohistochemistry and dshows Nissl staining. The grafts contained PF (FIG. 5A and FIG. 5D),PFAADC (FIG. 5B and FIG. 5E), or PFVMAA (FIG. 5C and FIG. 5F) cells. Thescale bar equals 500 μm.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

PD is a slowly progressive disease that affects the dopaminergic neuronsin the substantia nigra of the brain. Gradual degeneration of thesecells causes a reduction in dopamine. This decrease in dopamine canproduce one or more of the classic signs of PD. This neurologicalsyndrome is manifested by the combination of resting tremor (ortrembling) of the arms and legs, stiffness and rigidity, loss ofpostural reflexes, and slowness of movement. Other symptoms observed insome persons with PD can include, small cramped handwriting(micrographia); lack of arm swing on the affected side; decreased facialexpression (hypomimia); lowered voice volume (dysarthria); feelings ofdepression or anxiety; episodes of being “stuck in place” wheninitiating a step . . . called “freezing”; slight foot drag on theaffected side; increase in dandruff or oily skin; less frequent blinkingand swallowing.

There have been numerous attempts to treat PD patients. All suchattempts are based around the correction of the biochemical deficit thatmanifests in PD, i.e., the inability to supply dopamine. PD patientshave been routinely treated with a combination of L-DOPA and a DOPAdecarboxylase inhibitor such as carbidopa or benserazide. Unfortunately,patients develop fluctuating responses to such treatment after long termtherapy. Additional therapies have attempted to graft cells to the brainof affected patients. The source of these cells has primarily been thefetus. In most cases, providing such grafts merely increases the PDpatient's response to the L-DOPA provided as opposed to alleviating theneed for the drug. There are a few examples of patients who improveddramatically and did not require any medication. However, the difficultyin obtaining proper donor tissues in sufficient amounts renders clinicalapplication of this protocol premature.

Thus, short duration and fluctuating responses limit the efficacy ofL-DOPA therapy for PD. The loss of dopamine storage capacity is thoughtto be primarily responsible for development of such complications.Although PD cannot be prevented, individualized treatment may result inthe amelioration of the symptoms of PD, so that the afflicted individualcan live a complete and active life. The present invention is directedat methods and compositions for affecting such a beneficial outcome.

1. The Present Invention

The present invention circumvents the ethical and logistical problemsplaguing PD therapy by providing primary fibroblasts that have beengenetically modified to produce and store dopamine. Transplanting suchcells to a subject animal exhibiting PD restores normal behavior byproviding dopamine in an appropriate release from storage vesicles fromthe transplanted cells. More particularly, the inventor has engineeredprimary fibroblast cells to express an aromatic L-amino aciddecarboxylase in combination with a vesicular monoamine transporter-2 toproduce and store dopamine. Grafting these cells in the denervatedstriatum of Parkinsonian rats prolonged the duration of dopamineelevation and behavioral improvement after exogenous L-DOPAadministration.

The advantage of such a gene-based strategy over systemic, currentpharmacological methods is in part pharmacokinetics, ie., geneexpression is relatively stable leading to consistent production ofdopamine and stable striatal levels without the dose fluctuation andalterations in receptor sensitivity and signaling that are observed withoral dosing regimes. Another advantage is that the dopamine productionis confined to striatum, avoiding systemic and extrastriatal adverseeffects.

Thus, in a broad sense, the present invention provides a method forproducing dopamine in a cell. Additional embodiments contemplate methodsof treating and or alleviating the symptoms of PD as well as providingkits and compositions for use in such methods. These methods andcompositions are described in greater detail herein below.

a. Proteins Involved in Dopamine Production

The biochemical pathway for dopamine production is well characterized.Dopamine is a member of the catecholamine family of neurotransmitters.The catecholamines are derived biosynthetically form L-tyrosine andinclude dopamine, norephinephrine (noradrenaline) and ephinephrine(adrenaline).

The hydroxylation of L-tyrosine by tyrosine hydroxylase is the firstcommitted step in catecholamine biosynthesis. The action of thehydroxylase yields 3, 4,-dihydroxyphenylalanine (L-DOPA). L-DOPA issubsequently decarboxylated by an aromatic L-amino acid decarboxylase(AADC) to produce the human brain neurotransmitter dopamine.Noradrenaline is produced by the hydroxylation of dopamine andnoradrenaline is then rearranged to adrenaline. Once neurotransmittersare produced, neurotransmission depends on the regulated release ofchemical transmitters molecules. In order for this to happen, once thechemical are synthesized, they are packaged into specialized secretoryvesicles of neurons and neuroendocrine cells, a process that is mediatedby specific vesicular transporters. One such vesicular transporter isvesicular monoamine transporter. Thus, the principle enzymes involved inthe synthesis of dopamine are tyrosine hydroxylase and AADC. The presentsection provides a brief introduction to the proteins involved in thegeneration and transport of dopamine.

Aromatic L-Amino Acid Decarboxylase. In order for L-DOPA to be aneffective therapeutic for PD, it requires the action of AADC fordecarboxylation. This necessity of AADC for dopamine synthesis has beendemonstrated previously (Kang et al., 1993). The AADC enzyme has beenwell characterized from human and other mammalian tissues. Sequences forAADC can be found in Genbank, for example, Genbank Accession No.SEG_HUMAADC0; Genbank Accession No. U31884; Genbank Accession No.SEG_RATAADC; Genbank Accession No. M74029; Genbank Accession No.AF071068; Genbank Accession No. S82290; Genbank Accession No. Z11576;Genbank Accession No. SEG_RATAAADNN; Genbank Accession No. M76180;Genbank Accession No. M58049; Genbank Accession No. SEG_HUMARODE. Eachof these sequences is incorporated herein by reference as providingdisclosure of AADC protein. Of course it is understood that these aremerely exemplary sequences and that sequences variations may also beemployed. Methods of generating such variations are well known to thoseof skill in the art. Additionally, many other AADC proteins may be foundin the Genbank database that will be useful in the present invention.

Vesicular Monoamine Transporters. VMAT1 and VMAT 2 are two isoforms ofthe vesicular monoamine transporter initially identified using cDNAscreening strategies to investigate the passive and active uptakeprocesses in fibroblast cells (Erickson et al., 1992; Liu et al., 1992).Despite the fact that these cells are non-neuroendocrine in nature andlack synaptic vesicles, they do contain endosomes. The initial studiesin these cells now provide a convenient in vitro and in situ system forexpression and structure analysis of VMAT.

Two separate genes encode VMAT1 and VMAT2. The chromosomal locations ofthese two genes are 8p21 (Peter et al., 1993) and 10q26.1 (Erickson andEiden, 1993; Peter et al., 1993; Surratt et al., 1993), respectively.The existence of two genes for the VMAT isoforms suggests that they aredifferentially expressed in cell type sand impart functionally importantdifferences in vesicular monoamine storage in these cells. VMAT1 isfound in adrenal glands and is absent in brain tissue (Lui et al.,1992), whereas VMAT 2 is expressed in noradrenergic, dopaminergic andserotonergic cell bodies in the brain stem (Erickson et al., 1992; Luiet al., 1992). For the purposes of the present invention the vesicularmonoamine transporter will likely be VMAT2, however, it is contemplatedthat VMAT 1 also may be used.

Sequences for VMAT can be found in Genbank, for example, GenbankAccession No. U39905; Genbank Accession No. AF047575; Genbank AccessionNo. AA612554; Genbank Accession No. X76380; Genbank Accession No.X71354; Genbank Accession No. L00603; Genbank Accession No. U02876. Eachof these sequences is incorporated herein by reference as providingdisclosure of VMAT protein. Of course it is understood that these aremerely exemplary sequences and that sequences variations may also beemployed. Methods of generating such variations are well known to thoseof skill in the art.

The characteristics of vesicular monoamine transport by VMAT1 and VMAT2from rat, bovine and human cells have been extensively described in theliterature (Erickson et al., 1992; Erickson et al., 1996; Liu et al.,1992; Erickson and Eiden, 1993; Gasnier et al., 1994; Howell et al.,1994; Peter et al., 1994; Weihe et al., 1994). Uptake is energydependent and the biochemical characteristics of this uptake are welldocuments (Varoqui and Erickson, 1997).

The cytosolic concentrations of amines in monoaminergic neurons andendocrine cells is estimated to be approximately 10-20 μM (Phillips,1982; Silva and Bunney, 1988). It is envisioned that the cells of thepresent invention may be able to produce such concentrations. However,concentrations of about 2 μM dopamine, about 3 μM dopamine, about 4 μMdopamine, about 5 μM dopamine, about 6 μM dopamine, about 7 μM dopamine,about 8 μM dopamine, about 9 μM dopamine, about 11 μM dopamine, about 12μM dopamine, about 14 μM dopamine, about 16 μM dopamine, about 18 μMdopamine, about 22 μM dopamine, about 24 μM dopamine, about 26 μMdopamine, about 28 μM dopamine, about 30 μM dopamine, or more may beachieved. It is contemplated that the engineered cells of the presentinvention will secrete dopamine. The concentration of dopamine secretedmay vary between batches of cells but will be in the range of 120 pmolesdopamine/10⁶ cells/hour. This is only an exemplary figure and it iscontemplate that the rates of secretion may be 20 pmoles dopamine/10⁶cells/hour; 30 pmoles dopamine/10⁶ cells/hour; 40 pmoles dopamine/10⁶cells/hour; 50 pmoles dopamine/10⁶ cells/hour; 60 pmoles dopamine/10⁶cells/hour; 70 pmoles dopamine/10⁶ cells/hour; 80 pmoles dopamine/10⁶cells/hour; 90 pmoles dopamine/10⁶ cells/hour; 100 pmoles dopamine/10⁶cells/hour; 110 pmoles dopamine/10⁶ cells/hour; 120 pmoles dopamine/10⁶cells/hour; 130 pmoles dopamine/10⁶ cells/hour; 140 pmoles dopamine/10⁶cells/hour; 150 pmoles dopamine/10⁶ cells/hour; 160 pmoles dopamine/10⁶cells/hour; 170 pmoles dopamine/10⁶ cells/hour; 180 pmoles dopamine/10⁶cells/hour; 200 pmoles dopamine/10⁶ cells/hour; 220 pmoles dopamine/10⁶cells/hour; 240 pmoles dopamine/10⁶ cells/hour; 260 pmoles dopamine/10⁶cells/hour; 280 pmoles dopamine/10⁶ cells/hour; 300 pmoles dopamine/10⁶cells/hour; 320 pmoles dopamine/10⁶ cells/hour; 340 pmoles dopamine/10⁶cells/hour; 360 pmoles dopamine/10⁶ cells/hour; 380 pmoles dopamine/10⁶cells/hour; 400 pmoles dopamine/10⁶ cells/hour; 450 pmoles dopamine/10⁶cells/hour; 500 pmoles dopamine/10⁶ cells/hour; 650 pmoles dopamine/10⁶cells/hour; 700 pmoles dopamine/10⁶ cells/hour; 750 pmoles dopamine/10⁶cells/hour; 800 pmoles dopamine/10⁶ cells/hour; 850 pmoles dopamine/10⁶cells/hour; 900 pmoles dopamine/10⁶ cells/hour; 1000 pmoles dopamine/10⁶cells/hour. Of course it is contemplated that the cells may secreteranges between any of these figures and may even secrete more than 1000pmoles dopamine/10⁶ cells/hour.

A genetically modified population of cells in which the cells have beengenetically modified to express an AADC and a VMAT transgene thatsecretes between about 20 pmoles dopamine/10⁶ cells/hour and about 1000pmoles dopamine/10⁶ cells/hour is one population of cells that will beuseful in the present invention. A genetically modified population ofcells in which the cells have been genetically modified to express anAADC and a VMAT transgene that secretes between about 40 pmolesdopamine/10⁶ cells/hour and about 900 pmoles dopamine/10⁶ cells/hour isone population of cells that will be useful in the present invention. Agenetically modified population of cells in which the cells have beengenetically modified to express an AADC and a VMAT transgene thatsecretes between about 60 pmoles dopamine/10⁶ cells/hour and about 850pmoles dopamine/10⁶ cells/hour is one population of cells that will beuseful in the present invention. A genetically modified population ofcells in which the cells have been genetically modified to express anAADC and a VMAT transgene that secretes between about 80 pmolesdopamine/10⁶ cells/hour and about 800 pmoles dopamine/10⁶ cells/hour isone population of cells that will be useful in the present invention. Agenetically modified population of cells in which the cells have beengenetically modified to express an AADC and a VMAT transgene thatsecretes between about 100 pmoles dopamine/10⁶ cells/hour and about 700pmoles dopamine/10⁶ cells/hour is one population of cells that will beuseful in the present invention. A genetically modified population ofcells in which the cells have been genetically modified to express anAADC and a VMAT transgene that secretes between about 150 pmolesdopamine/10⁶ cells/hour and about 650 pmoles dopamine/10⁶ cells/hour isone population of cells that will be useful in the present invention. Agenetically modified population of cells in which the cells have beengenetically modified to express an AADC and a VMAT transgene thatsecretes between about 200 pmoles dopamine/10⁶ cells/hour and about 600pmoles dopamine/10⁶ cells/hour is one population of cells that will beuseful in the present invention. A genetically modified population ofcells in which the cells have been genetically modified to express anAADC and a VMAT transgene that secretes between about 250 pmolesdopamine/10⁶ cells/hour and about 500 pmoles dopamine/10⁶ cells/hour isone population of cells that will be useful in the present invention.Given the methods and compositions described in the present invention,it will be well within the skill of one in the art to producepopulations of cells having various other ranges of dopamine secretion.

The maturation time of monoaminergic vesicles, in terms of time requiredto reach steady-state vesicular monoamine accumulation is highlyvariable being approximately 2 to 4 minutes in the brain, 30 to 60minutes in the sympathetic nervous system and 30 to 60 hours in theadrenal medullae (Scherman and Boschi, 1988). The reasons for thesedifferences may relate to the presence of different classes of regulatedsecretory organelles expressing the transporter in endocrine versusneuronal cells.

Using the methods of the present invention, it will be possible to graftcells into subjects exhibiting PD symptoms. These cells will contain thetransgenes encoding AADC and a VMAT. The dopamine produced by the actionof the AADC will be stored in storage vesicles through the action of theVMAT. Thus the cells will store dopamine in appropriate concentrations,and subsequently release this dopamine in response to the appropriatesignals. It is contemplated that the cells may be “loaded” with dopaminebefore they are grafted into the patient, i.e., the synthesis andstorage of the dopamine is allowed to occur in vitro, prior totransplantation. Alternatively, the cells expressing the AADC and VMATtransgenes are transplanted into the appropriate area of the brain andsubsequently contacted with L-DOPA in order to affect the in vivoproduction of dopamine.

Tyrosine Hydroxylase. Tyrosine Hydroxylase is the rate limiting enzymein dopamine biosynthesis and is dramatically depleted in the putamen andcaudete of Parkinsonian patients and expression of TH and GTPcyclohydrolase I (Bencsics et al., 1996) in the stratium by either exvivo or in vivo strategies lead to recovery in animal models of thedisease. TH and GTP cyclohydrolase I transgene expression in striatalcells, either neuron and/or glia is essentially a biological L-DOPAtreatment. The sequence of tyrosine hydroxylase is well known to thoseof skill in the art. For example, Genbank accession No. AI114408,Y00414, X05290; SEG_HUMTHA0; AF070918; SEG_HUMTHIS0; SEG_HUMTH; D00269;AF030336; AJ000731; AF007942; X76209; Y00414; X05290; X53503; U14395;M17589; M24778. Each of these sequences is incorporated herein byreference as providing disclosure of TH protein. Of course it isunderstood that these are merely exemplary sequences and that sequencesvariations may also be employed. Methods of generating such variationsare well known to those of skill in the art. Additionally, many other THproteins may be found in the Genbank database that will be useful in thepresent invention.

In order to provide L-DOPA into the brain by gene therapy, most studieshave focused on the reintroducing TH, with or without GTP cyclohydrolaseI, either by direct transfer using viral vectors or by transplantinggenetically modified cells (Horellou et al., 1990; Fisher et al., 1991;During et al., 1994; Bencsics et al., 1996; Mandel et al., 1998). Incertain embodiments of the present invention, it is envisioned that THand GTP cyclohydrolase I may be combined with AADC and VMAT to achieve acomplete system for the synthesis and sequestering of dopamine.

b. Donor Cells

In any grafting technique, the choice of the donor cells forimplantation depends heavily on the nature of the expressed gene,characteristics of the vector and the desired phenotypic result. Forexample, seeing as retroviral vectors require cell division and DNAsynthesis for efficient infection, integration and gene expression(Weiss et al., 1985), if such vectors are used, the donor cells arepreferably actively growing cells, such as primary fibroblast culture orestablished cell lines, replicating embryonic neuronal cells orreplicating adult neuronal cells from selected areas such as theolfactory mucosa and possibly developing or reactive glia.

Primary cells, i.e. cells that have been freshly obtained from asubject, such as fibroblasts, that are not in the transformed state arepreferred for use in the present invention. Other suitable donor cellsinclude immortalized (transformed cells that continue to divide)fibroblasts, glial cells, adrenal cells, hippocampal cells,keratinocytes, hepatocytes, connective tissue cells, ependymal cells,bone marrow cells, stem cells, leukocytes, chromaffin cells and othermammalian cells susceptible to genetic manipulation and grafting usingthe methods of the present invention. Additional characteristics ofdonor cells which are relevant to successful grafting include the age ofthe donor cells.

Furthermore, there are available methods to induce a state ofsusceptibility in stationary, non-replicating target cells that willallow many other cell types to be suitable targets for viraltransduction. For instance, methods have been developed that permit thesuccessful viral vector infection of primary cultures of adult rathepatocytes, ordinarily refractory to infection with such vectors, andsimilar methods may be helpful for a number of other cells (Wolff etal., 1987). In addition, the development of many other kinds of vectorsderived from herpes, vaccinia, adenovirus, or other viruses, as well asthe use of efficient non-viral methods for introducing DNA into donorcells such as electroporation (Toneguzzo et al., 1986), lipofection ordirect gene insertion may be used for gene transfer into many othercells. These methods of gene transfer are discussed in further detailelsewhere in the specification.

c. L-DOPA

In particular aspects of the present invention, L-DOPA will be providedto an engineered cell of the present invention. Medicaments andcompositions of L-DOPA are well known to those of skill in the art. Suchcompositions are discussed below, this discussion is not intended to bean exhaustive treatise but rather it is intended to provide examples ofsuch compositions.

Injection of soluble esters of L-DOPA have been proposed as atherapeutic tool for stabilization of patients with severe motorfluctuations following chronic L-DOPA therapy. The L-DOPA methyl esteris one such ester that is likely to be useful in the present invention.U.S. Pat. No. 5,017,607; U.S. Pat. No. 4,826,875; U.S. Pat. No.4,873,263; U.S. Pat. No. 4,663,349; U.S. Pat. No. 4,771,073; Juncos, etal., (1987) and Cooper, et al., (1987) are incorporated herein byreference as providing disclosure of methods of producing andformulating L-DOPA methyl ester.

Another composition that would be effective in combination with themethod of the present invention is L-DOPA ethyl ester. L-DOPA ethylester is described in the literature as the hydrochloride salt. Theproduction of L-DOPA ethyl ester is comprehensively discussed in U.S.Pat. No. 5 ,607,969, incorporated herein by reference.

2. Polynucleotides

The present invention provides methods and compositions of engineeringcells to produce and store dopamine. In order to achieve theseobjectives, the present invention provides transgenes that express anenzyme capable of decarboxylating aromatic amino acids and a transgenethat expresses a protein that has vesicular neurotransmitter transportactivity. These transgenes comprise polynucleotides that encode AADC,VMAT or any other protein that may be useful in the production andstorage of dopamine. Thus these polynucleotides may be inserted into ahost cell to produce a donor cell containing that polynucleotide whichmay, in some cases, be capable of expressing the product of thatpolynucleotide. In addition to therapeutic considerations, cellsexpressing polynucleotides of the present invention may prove useful inthe context of screening for agents that repress, inhibit, interferewith, block, abrogate, or otherwise ameliorate the PD.

a. Polynucleotides Encoding AADC or VMAT

Polynucleotides used according to the present invention may encode anentire AADC or VMAT gene, a domain, or any other fragment of the proteinsequences. Exemplary AADC and VMAT sequences are well known to those ofskill in the art and can be found in Genbank as described above. One ofskill in the art is referred to the Genbank database(http://www.ncbi.nlm.nih.gov/Entrez) which contains various other AADCand VMAT sequences.

As used herein, the term “transgene” means a polynucleotide insertedinto a donor cell encoding an amino acid sequence corresponding to afunctional protein having some or all of the activity attributed to thatparticular protein. Such a protein may be a therapeutic protein that iscapable of exerting a therapeutic effect on cells of the CNS or having aregulatory effect on the expression of a function in the cells of theCNS.

The polynucleotide may be derived from genomic DNA, i.e., cloneddirectly from the genome of a particular organism. In preferredembodiments, however, the polynucleotide would comprise complementaryDNA (cDNA). Also contemplated is a cDNA plus a natural intron or anintron derived from another gene; such engineered molecules are sometimereferred to as “mini-genes.”

The term “cDNA” is intended to refer to DNA prepared using messenger RNA(mRNA) as template. The advantage of using a cDNA, as opposed to genomicDNA or DNA polymerized from a genomic, non- or partially-processed RNAtemplate, is that the cDNA primarily contains coding sequences of thecorresponding protein. There may be times when the full or partialgenomic sequence is preferred, such as where the non-coding regions arerequired for optimal expression.

The polynucleotide may be derived from genomic DNA, i.e., cloneddirectly from the genome of a particular organism. In preferredembodiments, however, the polynucleotide would comprise complementaryDNA (cDNA). Also contemplated is a cDNA plus a natural intron or anintron derived from another gene; such engineered molecules are sometimereferred to as “mini-genes.” At a minimum, these and otherpolynucleotides of the present invention may be used as molecular weightstandards in, for example, gel electrophoresis.

The term “cDNA” is intended to refer to DNA prepared using messenger RNA(mRNA) as template. The advantage of using a cDNA, as opposed to genomicDNA or DNA polymerized from a genomic, non- or partially-processed RNAtemplate, is that the cDNA primarily contains coding sequences of thecorresponding protein. There may be times when the full or partialgenomic sequence is preferred, such as where the non-coding regions arerequired for optimal expression or where non-coding regions such asintrons are to be targeted in an antisense strategy.

Naturally, the present invention also encompasses the use ofpolynucleotides that are complementary, or essentially complementary, toAADC and VMAT. Polynucleotide sequences that are “complementary” arethose that are capable of base-pairing according to the standardWatson-Crick complementary rules. As used herein, the term“complementary sequences” means polynucleotide sequences that aresubstantially complementary, as may be assessed by the same nucleotidecomparison set forth above, or as defined as being capable ofhybridizing to AADC or VMAT under relatively stringent conditions suchas those described herein. Such sequences may encode the entire AADC orVMAT protein, or functional or non-functional fragments thereof.

It also is contemplated that a given AADC or VMAT from a given speciesmay be represented by natural variants that have slightly differentnucleic acid sequences but, nonetheless, encode the same protein. Asused in this application, the term “a polynucleotide encoding” refers toa polynucleotide molecule that has been isolated free of total cellularnucleic acid. The term “functionally equivalent codon” is used herein torefer to codons that encode the same amino acid, such as the six codonsfor arginine or serine (Table 1, below), and also refers to codons thatencode biologically equivalent amino acids, as discussed in thefollowing pages.

TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys CUGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGAAGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr TACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

Allowing for the degeneracy of the genetic code, sequences that have atleast about 50%, usually at least about 60%, more usually about 70%,most usually about 80%, preferably at least about 90% and mostpreferably about 95% of nucleotides that are identical to AADC or VMATare polynucleotides that may be used in the present invention.Polynucleotides that are essentially the same as AADC or VMAT are alsofunctionally defined as sequences that are capable of hybridizing to apolynucleotide segment containing the complement of AADC or VMAT understandard conditions.

The polynucleotides of the present invention include those encodingbiologically functional equivalent AADC or VMAT proteins and peptides.Such sequences may arise as a consequence of codon redundancy and aminoacid functional equivalency that are known to occur naturally withinnucleic acid sequences and the proteins thus encoded. Alternatively,functionally equivalent proteins or peptides may be created via theapplication of recombinant DNA technology, in which changes in theprotein structure may be engineered, based on considerations of theproperties of the amino acids being exchanged. Changes designed byhumans may be introduced through the application of site-directedmutagenesis techniques or may be introduced randomly and screened laterfor the desired function, as described below.

b. Oligonucleotide Probes and Primers

In the present invention, an oligonucleotide probe or primer may beutilized which encodes a domain or fragment of AADC or VMAT. A AADC orVMAT probe or primer may be used in a number of molecular biologytechniques well known to those skilled in the art including, but notlimited to, Southern or Northern blot hybridization, in situhybridization, or polymerase chain reaction (PCR). One method ofexploiting probes and primers of the present invention is insite-directed, or site-specific mutagenesis.

Site-specific mutagenesis is a technique useful in the preparation ofindividual peptides, or biologically functional equivalent proteins orpeptides, through specific mutagenesis of the underlying DNA. Thetechnique further provides a ready ability to prepare and test sequencevariants, incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into the DNA.Site-specific mutagenesis allows for the production of mutants throughthe use of specific oligonucleotide sequences which encode the DNAsequence of the desired mutation, as well as a sufficient number ofadjacent nucleotides, to provide a primer sequence of sufficient sizeand sequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 17 to 25nucleotides in length is preferred, with about 5 to 10 residues on bothsides of the junction of the sequence being altered.

The technique typically employs a bacteriophage vector that exists inboth a single-stranded and double-stranded form. Typical vectors usefulin site-directed mutagenesis include vectors such as the M13 phage.These phage vectors are commercially available and their use isgenerally well known to those skilled in the art. Double-strandedplasmids are also routinely employed in site directed mutagenesis, whicheliminates the step of transferring the gene of interest from a phage toa plasmid.

In general, site-directed mutagenesis is performed by first obtaining asingle-stranded vector, or melting of two strands of a double-strandedvector which includes within its sequence a DNA sequence encoding thedesired protein. An oligonucleotide primer bearing the desired mutatedsequence is synthetically prepared. This primer is then annealed withthe single-stranded DNA preparation, taking into account the degree ofmismatch when selecting hybridization conditions, and subjected to DNApolymerizing enzymes such as E. coli polymerase I Klenow fragment, inorder to complete the synthesis of the mutation-bearing strand. Thus, aheteroduplex is formed wherein one strand encodes the originalnon-mutated sequence and the second strand bears the desired mutation.This heteroduplex vector is then used to transform appropriate cells,such as E. coli cells, and clones are selected that include recombinantvectors bearing the mutated sequence arrangement.

The preparation of sequence variants of the selected gene usingsite-directed mutagenesis is provided as a means of producingpotentially useful species and is not meant to be limiting, as there areother ways in which sequence variants of genes may be obtained. Forexample, recombinant vectors encoding the desired gene may be treatedwith mutagenic agents, such as hydroxylamine, to obtain sequencevariants.

It may be advantageous to combine portions of genomic DNA with cDNA orsynthetic sequences to generate specific constructs. For example, wherean intron is desired in the ultimate construct, a genomic clone willneed to be used. The cDNA or a synthesized polynucleotide may providemore convenient restriction sites for the remaining portion of theconstruct and, therefore, would be used for the rest of the sequence.

3. Polypeptides

According to the present invention, AADC or VMAT may act in concert toproduce and store dopamine in a genetically engineered cell. While thesemolecules are structurally distinct, they are part of a pathway involvedin the production and release of dopamine and other catecholamineneurotransmitters. In addition to the complete AADC or VMAT molecules,the present invention also relates to fragments of the polypeptides thatmay or may not retain their respective decarboxylase and transporteractivities. Fragments including the N-terminus of the molecule may begenerated by genetic engineering of translation stop sites within thecoding region. Alternatively, treatment of the AADC or VMAT moleculeswith proteolytic enzymes, known as protease, can produces a variety ofN-terminal, C-terminal and internal fragments. These fragments may bepurified according to known methods, such as precipitation (e.g,ammonium sulfate), HPLC, ion exchange chromatography, affinitychromatography (including immunoaffinity chromatography) or various sizeseparations (sedimentation, gel electrophoresis, gel filtration).

A specialized kind of insertional variant is the fusion protein. Thismolecule generally has all or a substantial portion of the nativemolecule, linked at the N- or C-terminus, to all or a portion of asecond polypeptide. For example, fusions typically employ leadersequences from other species to permit the recombinant expression of aprotein in a heterologous host. Another useful fusion includes theaddition of a immunologically active domain, such as an antibodyepitope, to facilitate purification of the fusion protein. Inclusion ofa cleavage site at or near the fusion junction will facilitate removalof the extraneous polypeptide after purification. Other useful fusionsinclude linking of functional domains, such as active sites fromenzymes, glycosylation domains, cellular targeting signals ortransmembrane regions. One particular fusion of interest would includeall or a portion of the native AADC molecule, linked at the N- orC-terminus, to all or a portion of a VMAT polypeptide.

4. Vectors for Cloning, Gene Transfer and Expression

Within certain embodiments, expression vectors are employed to introducethe AADC or VMAT into the host or donor cell. Expression requires thatappropriate signals be provided in the vectors, which include variousregulatory elements, such as enhancers/promoters from both viral andmammalian sources that drive expression of the genes of interest in hostcells. Elements designed to optimize messenger RNA stability andtranslatability in host cells are also defined. The conditions for theuse of a number of dominant drug selection markers for establishingpermanent, stable cell clones expressing the products are also provided,as is an element that links expression of the drug selection markers toexpression of the polypeptide.

a. Regulatory Elements

Throughout this application, the term “expression construct” is meant toinclude any type of genetic construct containing a polynucleotide codingfor a gene product in which part or all of the polynucleotide encodingsequence is capable of being transcribed. The transcript may betranslated into a protein, but it need not be. In certain embodiments,expression includes both transcription of a gene and translation of mRNAinto a gene product. In other embodiments, expression only includestranscription of the polynucleotide encoding a gene of interest.

In preferred embodiments, the polynucleotide encoding a gene product isunder transcriptional control of a promoter. A “promoter” refers to aDNA sequence recognized by the synthetic machinery of the cell, orintroduced synthetic machinery, required to initiate the specifictranscription of a gene. The phrase “under transcriptional control”means that the promoter is in the correct location and orientation inrelation to the polynucleotide to control RNA polymerase initiation andexpression of the gene.

The term promoter will be used here to refer to a group oftranscriptional control modules that are clustered around the initiationsite for RNA polymerase II. Much of the thinking about how promoters areorganized derives from analyses of several viral promoters, includingthose for the HSV thymidine kinase (tk) and SV40 early transcriptionunits. These studies, augmented by more recent work, have shown thatpromoters are composed of discrete functional modules, each consistingof approximately 7-20 bp of DNA, and containing one or more recognitionsites for transcriptional activator or repressor proteins.

At least one module in each promoter functions to position the startsite for RNA synthesis. The best known example of this is the TATA box,but in some promoters lacking a TATA box, such as the promoter for themammalian terminal deoxynucleotidyl transferase gene and the promoterfor the SV40 late genes, a discrete element overlying the start siteitself helps to fix the place of initiation.

Additional promoter elements regulate the frequency of transcriptionalinitiation. Typically, these are located in the region 30-110 bpupstream of the start site, although a number of promoters have recentlybeen shown to contain functional elements downstream of the start siteas well. The spacing between promoter elements frequently is flexible,so that promoter function is preserved when elements are inverted ormoved relative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 bp apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can function either co-operatively or independently to activatetranscription.

The particular promoter employed to control the expression of apolynucleotide sequence of interest is not believed to be important, solong as it is capable of directing the expression of the polynucleotidein the targeted cell. Thus, where a human cell is targeted, it ispreferable to position the polynucleotide coding region adjacent to andunder the control of a promoter that is capable of being expressed in ahuman cell. Generally speaking, such a promoter might include either ahuman or viral promoter.

In various embodiments, the human cytomegalovirus (CMV) immediate earlygene promoter, the SV40 early promoter, the Rous sarcoma virus longterminal repeat, rat insulin promoter and glyceraldehyde-3-phosphatedehydrogenase can be used to obtain high-level expression of the codingsequence of interest. The use of other viral or mammalian cellular orbacterial phage promoters which are well-known in the art to achieveexpression of a coding sequence of interest is contemplated as well,provided that the levels of expression are sufficient for a givenpurpose. By employing a promoter with well-known properties, the leveland pattern of expression of the protein of interest followingtransfection or transformation can be optimized.

Selection of a promoter that is regulated in response to specificphysiologic or synthetic signals can permit inducible expression of thegene product. For example in the case where expression of a transgene,or transgenes when a multicistronic vector is utilized, is toxic to thecells in which the vector is produced in, it may be desirable toprohibit or reduce expression of one or more of the transgenes. Examplesof transgenes that may be toxic to the producer cell line arepro-apoptotic and cytokine genes. Several inducible promoter systems areavailable for production of viral vectors where the transgene productmay be toxic.

The ecdysone system (Invitrogen, Carlsbad, Calif.) is one such system.This system is designed to allow regulated expression of a gene ofinterest in mammalian cells. It consists of a tightly regulatedexpression mechanism that allows virtually no basal level expression ofthe transgene, but over 200-fold inducibility. The system is based onthe heterodimeric ecdysone receptor of Drosophila, and when ecdysone oran analog such as muristerone A binds to the receptor, the receptoractivates a promoter to turn on expression of the downstream transgenehigh levels of mRNA transcripts are attained. In this system, bothmonomers of the heterodimeric receptor are constitutively expressed fromone vector, whereas the ecdysone-responsive promoter which drivesexpression of the gene of interest is on another plasmid. Engineering ofthis type of system into the gene transfer vector of interest wouldtherefore be useful. Cotransfection of plasmids containing the gene ofinterest and the receptor monomers in the producer cell line would thenallow for the production of the gene transfer vector without expressionof a potentially toxic transgene. At the appropriate time, expression ofthe transgene could be activated with ecdysone or muristeron A.

Another inducible system that would be useful is the Tet-Off™ or Tet-On™system (Clontech, Palo Alto, Calif.) originally developed by Gossen andBujard (Gossen and Bujard, 1992; Gossen et al., 1995). This system alsoallows high levels of gene expression to be regulated in response totetracycline or tetracycline derivatives such as doxycycline. In theTet-On™ system, gene expression is turned on in the presence ofdoxycycline, whereas in the Tet-Off™ system, gene expression is turnedon in the absence of doxycycline. These systems are based on tworegulatory elements derived from the tetracycline resistance operon ofE. coli. The tetracycline operator sequence to which the tetracyclinerepressor binds, and the tetracycline repressor protein. The gene ofinterest is cloned into a plasmid behind a promoter that hastetracycline-responsive elements present in it. A second plasmidcontains a regulatory element called the tetracycline-controlledtransactivator, which is composed, in the Tet-Off™ system, of the VP16domain from the herpes simplex virus and the wild-type tertracyclinerepressor. Thus in the absence of doxycycline, transcription isconstitutively on. In the Tet-On™ system, the tetracycline repressor isnot wild type and in the presence of doxycycline activatestranscription. For gene therapy vector production, the Tet-Off™ systemwould be preferable so that the producer cells could be grown in thepresence of tetracycline or doxycycline and prevent expression of apotentially toxic transgene, but when the vector is introduced to thepatient, the gene expression would be constitutively on.

In some circumstances, it may be desirable to regulate expression of atransgene in a gene therapy vector. For example, different viralpromoters with varying strengths of activity may be utilized dependingon the level of expression desired. In mammalian cells, the CMVimmediate early promoter if often used to provide strong transcriptionalactivation. Modified versions of the CMV promoter that are less potenthave also been used when reduced levels of expression of the transgeneare desired. When expression of a transgene in hematopoetic cells isdesired, retroviral promoters such as the LTRs from MLV or MMTV areoften used. Other viral promoters that may be used depending on thedesired effect include SV40, RSV LTR, HIV-1 and HIV-2 LTR, adenoviruspromoters such as from the E1A, E2A, or MLP region, AAV LTR, cauliflowermosaic virus, HSV-TK, and avian sarcoma virus.

Similarly tissue specific promoters may be used to effect transcriptionin specific tissues or cells so as to reduce potential toxicity orundesirable effects to non-targeted tissues. For example, promoters suchas the PSA, probasin, prostatic acid phosphatase or prostate-specificglandular kallikrein (hK2) may be used to target gene expression in theprostate.

More particularly, it is contemplated that promoters specific for VMATand/or AADC as well as TH may be used in the present invention. Suchpromoters are well known to those of skill in the art. For example,Genbank Accession No. HUMTHC discloses a human TH promoter region,Genbank Accession No. S74903 discloses an AADC non-neuronal promoter;Genbank Accession No. AF023677 discloses a TH promoter; GenbankAccession No. M23597 discloses a tyrosine hydroxylase promoter region;Genbank Accession No. L05074 discloses an AADC promoter region andGenbank Accession No. AF047575 discloses a VMAT2 promoter region. Ofcourse these are only exemplary promoters and any genomic sequences forAADC, VMAT or TH coding genes will contain promoter regions specific forthose genes. Genbank Accession Nos. L29478, Z30952 disclose human GTPcyclohydrolase I genes, the promoter region(s) from this and other GTPcyclohydrolase I genes will be particularly useful in the presentinvention. More particularly, Genbank Accession No. D38603 discloses thepromoter region from a H. sapiens GTP cyclohydrolase I gene. Anotheruseful promoter will be the promoter from the gene encoding dopaminetransporter (see for example the gene sequences disclosed in GenbankAccession Nos. AF079899; U92262; SEG_HSDOPTRP; U16265; SEG_HUMDATS, eachincorporated herein by reference).

In certain indications, it may be desirable to activate transcription atspecific times after administration of the gene therapy vector. This maybe done with such promoters as those that are hormone or cytokineregulatable. For example in gene therapy applications where theindication is a gonadal tissue where specific steroids are produced orrouted to, use of androgen or estrogen regulated promoters may beadvantageous. Such promoters that are hormone regulatable include MMTV,MT-1, ecdysone and RuBisco. Other hormone regulated promoters such asthose responsive to thyroid, pituitary and adrenal hormones are expectedto be useful in the present invention. Cytokine and inflammatory proteinresponsive promoters that could be used include K and T Kininogen(Kageyama et al., 1987), c-fos, TNF-alpha, C-reactive protein (Arcone etal., 1988), haptoglobin (Oliviero et al., 1987), serum amyloid A2, C/EBPalpha, IL-1, IL-6 (Poli and Cortese, 1989), Complement C3 (Wilson etal., 1990), IL-8, alpha-1 acid glycoprotein (Prowse and Baumann, 1988),alpha-1 antitypsin, lipoprotein lipase (Zechner et al., 1988),angiotensinogen (Ron et al., 1991), fibrinogen, c-jun (inducible byphorbol esters, TNF-alpha, UV radiation, retinoic acid, and hydrogenperoxide), collagenase (induced by phorbol esters and retinoic acid),metallothionein (heavy metal and glucocorticoid inducible), Stromelysin(inducible by phorbol ester, interleukin-I and EGF), alpha-2macroglobulin and alpha-1 antichymotrypsin.

Tumor specific promoters such as osteocalcin, hypoxia-responsive element(HRE), MAGE-4, CEA, alpha-fetoprotein, GRP78/BiP and tyrosinase may alsobe used to regulate gene expression in tumor cells. Other promoters thatcould be used according to the present invention includeLac-regulatable, chemotherapy inducible (e.g. MDR), and heat(hyperthermia) inducible promoters, radiation-inducible (e.g., EGR (Jokiet al., 1995)), Alpha-inhibin, RNA pol III tRNA met and other amino acidpromoters, U1 snRNA (Bartlett et al., 1996), MC-1, PGK, β-actin andα-globin. Many other promoters that may be useful are listed in Waltherand Stein (1996).

Enhancers are genetic elements that increase transcription from apromoter located at a distant position on the same molecule of DNA.Enhancers are organized much like promoters. That is, they are composedof many individual elements, each of which binds to one or moretranscriptional proteins. The basic distinction between enhancers andpromoters is operational. An enhancer region as a whole must be able tostimulate transcription at a distance; this need not be true of apromoter region or its component elements. On the other hand, a promotermust have one or more elements that direct initiation of RNA synthesisat a particular site and in a particular orientation, whereas enhancerslack these specificities. Promoters and enhancers are frequentlyoverlapping and contiguous, often seeming to have a very similar modularorganization.

Where a cDNA insert is employed, one will typically desire to include apolyadenylation signal to effect proper polyadenylation of the genetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and any suchsequence may be employed such as human growth hormone and SV40polyadenylation signals. Also contemplated as an element of theexpression cassette is a terminator. These elements can serve to enhancemessage levels and to minimize read through from the cassette into othersequences.

b. Selectable Markers

In certain embodiments of the invention, the cells containpolynucleotide constructs of the present invention, and a cell may beidentified in vitro or in vivo by including a marker in the expressionconstruct. Such markers will confer an identifiable change to the cellpermitting easy identification of cells containing the expressionconstruct. Usually the inclusion of a drug selection marker aids incloning and in the selection of transformants, for example, genes thatencode neomycin, puromycin, hygromycin, DHFR, GPT, HPRT, zeocin, andhistidinol are useful selectable markers. Alternatively, enzymes such asherpes simplex virus thymidine kinase (tk) or chloramphenicolacetyltransferase (CAT) may be employed. Immunologic markers also can beemployed. The selectable marker employed is not believed to beimportant, so long as it is capable of being expressed simultaneouslywith the polynucleotide encoding a gene product. Further examples ofselectable markers are well known to one of skill in the art.

c. Multigene Constructs and IRES

In certain embodiments of the invention, internal ribosome binding sites(IRES) elements are used to create multigene, or polycistronic,messages. IRES elements are able to bypass the ribosome scanning modelof 5′ methylated Cap dependent translation and begin translation atinternal sites (Pelletier and Sonenberg, 1988). IRES elements from twomembers of the picanovirus family (polio and encephalomyocarditis) havebeen described (Pelletier and Sonenberg, 1988), as well an IRES from amammalian message (Macejak and Sarnow, 1991). IRES elements can belinked to heterologous open reading frames. Multiple open reading framescan be transcribed together, each separated by an IRES, creatingpolycistronic messages. By virtue of the IRES element, each open readingframe is accessible to ribosomes for efficient translation. Multiplegenes can be efficiently expressed using a single promoter/enhancer totranscribe a single message.

Any heterologous open reading frame can be linked to IRES elements. Thisincludes genes for secreted proteins, multi-subunit proteins, encoded byindependent genes, intracellular or membrane-bound proteins, andselectable markers. In this way, expression of several proteins can besimultaneously engineered into a cell with a single construct and asingle selectable marker.

5. Introduction of Transgene into a Host Cell

There are a number of ways to introduce expression vectors into cells.In certain embodiments of the invention, the expression constructcomprises a virus or engineered construct derived from a viral genome.The ability of certain viruses to enter cells via receptor-mediatedendocytosis, to integrate into host cell genome and express viral genesstably and efficiently have made them attractive candidates for thetransfer of foreign genes into mammalian cells (Ridgeway, 1988; Nicolasand Rubenstein, 1988; Baichwal and Sugden, 1986; Temin, 1986). The firstviruses used as gene vectors were DNA viruses including thepapovaviruses (simian virus 40, bovine papilloma virus, and polyoma)(Ridgeway, 1988; Baichwal and Sugden, 1986) and adenoviruses (Ridgeway,1988; Baichwal and Sugden, 1986). These have a relatively low capacityfor foreign DNA sequences and have a restricted host spectrum.Furthermore, their oncogenic potential and cytopathic effects inpermissive cells raise safety concerns. They can accommodate only up to8 kb of foreign genetic material but can be readily introduced in avariety of cell lines and laboratory animals (Nicolas and Rubenstein,1988; Temin, 1986).

a. Viral Delivery

Retroviral Vectors. The retroviruses are a group of single-stranded RNAviruses characterized by an ability to convert their RNA todouble-stranded DNA in infected cells by a process ofreverse-transcription (Coffin, 1990). The resulting DNA then stablyintegrates into cellular chromosomes as a provirus and directs synthesisof viral proteins. The integration results in the retention of the viralgene sequences in the recipient cell and its descendants. The retroviralgenome contains three genes, gag, pol, and env that code for capsidproteins, polymerase enzyme, and envelope components, respectively. Asequence found upstream from the gag gene contains a signal forpackaging of the genome into virions. Two long terminal repeat (LTR)sequences are present at the 5′ and 3′ ends of the viral genome. Thesecontain strong promoter and enhancer sequences and are also required forintegration in the host cell genome (Coffin, 1990).

In order to construct a retroviral vector, a polynucleotide encoding agene of interest is inserted into the viral genome in the place ofcertain viral sequences to produce a virus that isreplication-defective. In order to produce virions, a packaging cellline containing the gag, pol, and env genes but without the LTR andpackaging components is constructed (Mann et al., 1983). When arecombinant plasmid containing a cDNA, together with the retroviral LTRand packaging sequences is introduced into this cell line (by calciumphosphate precipitation for example), the packaging sequence allows theRNA transcript of the recombinant plasmid to be packaged into viralparticles, which are then secreted into the culture media (Nicolas andRubenstein, 1988; Temin, 1986; Mann et al., 1983). The media containingthe recombinant retroviruses is then collected, optionally concentrated,and used for gene transfer. Retroviral vectors are able to infect abroad variety of cell types. However, integration and stable expressionrequire the division of host cells (Paskind et al., 1975).

A novel approach designed to allow specific targeting of retrovirusvectors was recently developed based on the chemical modification of aretrovirus by the chemical addition of lactose residues to the viralenvelope. This modification could permit the specific infection ofhepatocytes via sialoglycoprotein receptors.

A different approach to targeting of recombinant retroviruses wasdesigned in which biotinylated antibodies against a retroviral envelopeprotein and against a specific cell receptor were used. The antibodieswere coupled via the biotin components by using streptavidin (Roux etal., 1989). Using antibodies against major histocompatibility complexclass I and class II antigens, they demonstrated the infection of avariety of human cells that bore those surface antigens with anecotropic virus in vitro (Roux et al., 1989).

There are certain limitations to the use of retrovirus vectors in allaspects of the present invention. For example, retrovirus vectorsusually integrate into random sites in the cell genome. This can lead toinsertional mutagenesis through the interruption of host genes orthrough the insertion of viral regulatory sequences that can interferewith the function of flanking genes (Varmus et al., 1981). Anotherconcern with the use of defective retrovirus vectors is the potentialappearance of wild-type replication-competent virus in the packagingcells. This can result from recombination events in which theintact-sequence from the recombinant virus inserts upstream from thegag, pol, env sequence integrated in the host cell genome. However, newpackaging cell lines are now available that should greatly decrease thelikelihood of recombination (Markowitz et al., 1988; Hersdorffer et al.,1990).

Lentiviruses can also be used as vectors in the present application. Inaddition to the long-term expression of the transgene provided by allretroviral vectors, lentiviruses present the opportunity to transducenondividing cells and potentially achieve regulated expression. Thedevelopment of lentiviral vectors requires the design of transfervectors to ferry the transgene with efficient encapsidation of thetransgene RNA and with full expression capability, and of a packagingvector to provide packaging machinery in trans but without helper virusproduction. For both vectors, a knowledge of packaging signal isrequired—the signal to be included in the transfer vector but excludedfrom the packaging vector. Exemplary human lentiviruses are humanimmunodeficiency virus type 1 and type 2 (HIV-1 and HIV-2). HIV-2 islikely better suited for gene transfer than HIV-1 as it is lesspathogenic and thus safer during design and production; its desirablenuclear import and undesirable cell-cycle arrest functions aresegregated on two separate genes (Arya et al., 1998; Blomer et al.,1997).

Adenoviral Vectors. One of the preferred methods for in vivo deliveryinvolves the use of an adenovirus expression vector. “Adenovirusexpression vector” is meant to include those constructs containingadenovirus sequences sufficient to (a) support packaging of theconstruct and (b) to express an antisense polynucleotide that has beencloned therein. In this context, expression does not require that thegene product be synthesized.

The expression vector comprises a genetically engineered form ofadenovirus. Knowledge of the genetic organization of adenovirus, a 36kb, linear, double-stranded DNA virus, allows substitution of largepieces of adenoviral DNA with foreign sequences up to 7 kb (Grunhaus andHorwitz, 1992). In contrast to retrovirus, the adenoviral infection ofhost cells does not result in chromosomal integration because adenoviralDNA can replicate in an episomal manner without potential genotoxicity.Also, adenoviruses are structurally stable, and no genome rearrangementhas been detected after extensive amplification. Adenovirus can infectvirtually all epithelial cells regardless of their cell cycle stage. Sofar, adenoviral infection appears to be linked only to mild disease suchas acute respiratory disease in humans.

Adenovirus is particularly suitable for use as a gene transfer vectorbecause of its mid-sized genome, ease of manipulation, high titer, widetarget cell range and high infectivity. Both ends of the viral genomecontain 100-200 base pair inverted repeats (ITRs), which are ciselements necessary for viral DNA replication and packaging. The early(E) and late (L) regions of the genome contain different transcriptionunits that are divided by the onset of viral DNA replication. The E1region (E1A and E1B) encodes proteins responsible for the regulation oftranscription of the viral genome and a few cellular genes. Theexpression of the E2 region (E2A and E2B) results in the synthesis ofthe proteins for viral DNA replication. These proteins are involved inDNA replication, late gene expression and host cell shut-off (Renan,1990). The products of the late genes, including the majority of theviral capsid proteins, are expressed only after significant processingof a single primary transcript issued by the major late promoter (MLP).The MLP, (located at 16.8 m.u.) is particularly efficient during thelate phase of infection, and all the mRNAs issued from this promoterpossess a 5′-tripartite leader (TPL) sequence which makes them preferredmRNAs for translation.

In a current system, recombinant adenovirus is generated from homologousrecombination between shuttle vector and provirus vector. Due to thepossible recombination between two proviral vectors, wild-typeadenovirus may be generated from this process. Therefore, it is criticalto isolate a single clone of virus from an individual plaque and examineits genomic structure.

Generation and propagation of the current adenovirus vectors, which arereplication deficient, depend on a unique helper cell line, designated293, which was transformed from human embryonic kidney cells by Ad5 DNAfragments and constitutively expresses E1 proteins (Graham et al.,1977). Since the E3 region is dispensable from the adenovirus genome(Jones and Shenk, 1978), the current adenovirus vectors, with the helpof 293 cells, carry foreign DNA in either the E1, the D3 or both regions(Graham and Prevec, 1991). In nature, adenovirus can packageapproximately 105% of the wild-type genome (Ghosh-Choudhury et al.,1987), providing capacity for about 2 extra kb of DNA. Combined with theapproximately 5.5 kb of DNA that is replaceable in the E1 and E3regions, the maximum capacity of the current adenovirus vector is under7.5 kb, or about 15% of the total length of the vector. More than 80% ofthe adenovirus viral genome remains in the vector backbone and is thesource of vector-borne cytotoxicity. Also, the replication deficiency ofthe E1-deleted virus is incomplete. For example, leakage of viral geneexpression has been observed with the currently available vectors athigh multiplicities of infection (MOI) (Mulligan, 1993).

Helper cell lines may be derived from human cells such as humanembryonic kidney cells, muscle cells, hematopoietic cells or other humanembryonic mesenchymal or epithelial cells. Alternatively, the helpercells may be derived from the cells of other mammalian species that arepermissive for human adenovirus. Such cells include, e.g., Vero cells orother monkey embryonic mesenchymal or epithelial cells. As stated above,the preferred helper cell line is 293.

Recently, Racher et al. (1995) disclosed improved methods for culturing293 cells and propagating adenovirus. In one format, natural cellaggregates are grown by inoculating individual cells into 1 litersiliconized spinner flasks (Techne, Cambridge, UK) containing 100-200 mlof medium. Following stirring at 40 rpm, the cell viability is estimatedwith trypan blue. In another format, Fibra-Cel microcarriers (BibbySterlin, Stone, UK) (5 g/l) is employed as follows. A cell inoculum,resuspended in 5 ml of medium, is added to the carrier (50 ml) in a 250ml Erlenmeyer flask and left stationary, with occasional agitation, for1 to 4 hours. The medium is then replaced with 50 ml of fresh medium andshaking initiated. For virus production, cells are allowed to grow toabout 80% confluence, after which time the medium is replaced (to 25% ofthe final volume) and adenovirus added at an MOI of 0.05. Cultures areleft stationary overnight, following which the volume is increased to100% and shaking commenced for another 72 hours.

Other than the requirement that the adenovirus vector be replicationdefective, or at least conditionally defective, the nature of theadenovirus vector is not believed to be crucial to the successfulpractice of the invention. The adenovirus may be of any of the 42different known serotypes or subgroups A-F. Adenovirus type 5 ofsubgroup C is the preferred starting material in order to obtain theconditional replication-defective adenovirus vector for use in thepresent invention. This is because Adenovirus type 5 is a humanadenovirus about which a great deal of biochemical and geneticinformation is known, and it has historically been used for mostconstructions employing adenovirus as a vector.

As stated above, the typical vector according to the present inventionis replication defective and will not have an adenovirus E1 region.Thus, it will be most convenient to introduce the polynucleotideencoding the gene of interest at the position from which the E1-codingsequences have been removed. However, the position of insertion of theconstruct within the adenovirus sequences is not critical to theinvention. The polynucleotide encoding the gene of interest may also beinserted in lieu of the deleted E3 region in E3 replacement vectors asdescribed by Karlsson et al. (1986) or in the E4 region where a helpercell line or helper virus complements the E4 defect.

Adenovirus is easy to grow and manipulate and exhibits broad host rangein vitro and in vivo. This group of viruses can be obtained in hightiters, e.g., 10⁹-10¹¹ plaque-forming units per ml, and they are highlyinfective. The life cycle of adenovirus does not require integrationinto the host cell genome. The foreign genes delivered by adenovirusvectors are episomal and, therefore, have low genotoxicity to hostcells. No side effects have been reported in studies of vaccination withwild-type adenovirus (Couch et al., 1963; Top et al., 1971),demonstrating their safety and therapeutic potential as in vivo genetransfer vectors.

Adenovirus vectors have been used in eukaryotic gene expression (Levreroet al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhausand Horwitz, 1992; Graham and Prevec, 1992). Recently, animal studiessuggested that recombinant adenovirus could be used for gene therapy(Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet etal., 1990; Rich et al., 1993). Studies in administering recombinantadenovirus to different tissues include trachea instillation (Rosenfeldet al., 1991; Rosenfeld et al., 1992), muscle injection (Ragot et al.,1993), peripheral intravenous injections (Herz and Gerard, 1993) andstereotactic inoculation into the brain (Le Gal La Salle et al., 1993).

Adeno-associated Viral Vectors. Adeno-associated virus (AAV) utilizes alinear, single-stranded DNA of about 4700 base pairs. Inverted terminalrepeats flank the genome. Two genes are present within the genome,giving rise to a number of distinct gene products. The first, the capgene, produces three different virion proteins (VP), designated VP-1,VP-2 and VP-3. The second, the rep gene, encodes four non-structuralproteins (NS). One or more of these rep gene products is responsible fortransactivating AAV transcription.

The three promoters in AAV are designated by their location, in mapunits, in the genome. These are, from left to right, p5, pl9 and p40.Transcription gives rise to six transcripts, two initiated at each ofthree promoters, with one of each pair being spliced. The splice site,derived from map units 42-46, is the same for each transcript. The fournon-structural proteins apparently are derived from the longer of thetranscripts, and three virion proteins all arise from the smallesttranscript.

AAV is not associated with any pathologic state in humans.Interestingly, for efficient replication, AAV requires “helping”functions from viruses such as herpes simplex virus I and II,cytomegalovirus, pseudorabies virus and, of course, adenovirus. The bestcharacterized of the helpers is adenovirus, and many “early” functionsfor this virus have been shown to assist with AAV replication. Low levelexpression of AAV rep proteins is believed to hold AAV structuralexpression in check, and helper virus infection is thought to removethis block.

The terminal repeats of an AAV vector can be obtained by restrictionendonuclease digestion of AAV or a plasmid such as psub201, whichcontains a modified AAV genome (Samulski et al. 1987), or by othermethods known to the skilled artisan, including but not limited tochemical or enzymatic synthesis of the terminal repeats based upon thepublished sequence of AAV. The ordinarily skilled artisan can determine,by well-known methods such as deletion analysis, the minimum sequence orpart of the AAV ITRs which is required to allow function, i.e. stableand site-specific integration. The ordinarily skilled artisan also candetermine which minor modifications of the sequence can be toleratedwhile maintaining the ability of the terminal repeats to direct stable,site-specific integration.

AAV-based vectors have proven to be safe and effective vehicle for genedelivery in vitro, and these vectors are now being developed and testedin pre-clinical and clinical stages for a wide range of applications inpotential gene therapy, both ex vivo and in vivo. However, widevariations in AAV transduction efficiency in different cells and tissuesin vitro as well as in vivo has been repeatedly observed (Ponnazhagan etal., 1997b; 1997c; 1997d; 1997d) and others (Carter and Flotte, 1996;Chatterjee et al., 1995; Ferrari et al., 1996; Fisher et al, 1996;Flotte et al., 1993; Goodman et al., 1994; Kaplitt et al., 1994; 1996,Kessler et al., 1996; Koeberl et al., 1997; Mizukami et al., 1996; Xiaoet al., 1996).

AAV-mediated efficient gene transfer and expression in the lung has ledto clinical trials for the treatment of cystic fibrosis (Carter andFlotte, 1996; Flotte et al., 1993). Similarly, the prospects fortreatment of muscular dystrophy by AAV-mediated gene delivery of thedystrophin gene to skeletal muscle, of Parkinson's disease by tyrosinehydroxylase gene delivery to the brain, of hemophilia B by Factor IXgene delivery to the liver, and potentially of myocardial infarction byvascular endothelial growth factor gene to the heart, appear promisingsince AAV-mediated transgene expression in these organs has recentlybeen shown to be highly efficient (Fisher et al., 1996; Flotte et al.,1993; Kaplitt et al., 1994; 1996; Koeberl et al., 1997; McCown et al.,1996; Ping et al., 1996; Xiao et al., 1996).

Other Viral Vectors. Other viral vectors may be employed as expressionconstructs in the present invention. Vectors derived from viruses suchas vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar etal., 1988) adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal andSugden, 1986; Hermonat and Muzycska, 1984) moloney murine leukemia virus(MoMuLV); VSV-G type retroviruses (U.S. Pat. No. 5,817,491, specificallyincorporated herein by reference), papovaviruses such as JC, SV40,polyoma (U.S. Pat. No. 5,624,820, specifically incorporated herein byreference) Epstein-Barr Virus (EBV); papilloma viruses (U.S. Pat. No.5,674,703, specifically incorporated herein by reference), and moreparticularly, bovine papilloma virus type I (BPV; U.S. Pat. No.4,419,446, incorporated herein by reference); poliovirus herpesvirusesand other human and animal viruses may be employed. These viruses offerseveral attractive features for various mammalian cells (Friedmann,1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988;Horwich et al., 1990).

With the recent recognition of defective hepatitis B viruses, newinsight was gained into the structure-function relationship of differentviral sequences. In vitro studies showed that the virus could retain theability for helper-dependent packaging and reverse transcription despitethe deletion of up to 80% of its genome (Horwich et al., 1990). Thissuggested that large portions of the genome could be replaced withforeign genetic material. The hepatotropism and persistence(integration) were particularly attractive properties for liver-directedgene transfer. Chang et al. (1991) recently introduced thechloramphenicol acetyltransferase (CAT) gene into duck hepatitis B virusgenome in the place of the polymerase, surface, and pre-surface codingsequences. It was co-transfected with wild-type virus into an avianhepatoma cell line. Culture media containing high titers of therecombinant virus were used to infect primary duckling hepatocytes.Stable CAT gene expression was detected for at least 24 days aftertransfection (Chang et al., 1991).

In order to effect expression of sense or antisense gene constructs, theexpression construct must be delivered into a cell. This delivery may beaccomplished in vitro, as in laboratory procedures for transformingcells lines, or in vivo or ex vivo, as in the treatment of certaindisease states. One mechanism for delivery is via viral infection wherethe expression construct is encapsulated in an infectious viralparticle.

b. Non-viral Delivery

Several non-viral methods for the transfer of expression constructs intocultured mammalian cells also are contemplated by the present invention.These include calcium phosphate precipitation (Graham and Van Der Eb,1973; Chen and Okayama, 1987; Rippe et al., 1990) DEAE-dextran (Gopal,1985), electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984),direct microinjection (Harland and Weintraub, 1985), DNA-loadedliposomes (Nicolau and Sene, 1982; Fraley et al., 1979) andlipofectamine-DNA complexes, cell sonication (Fechheimer et al., 1987),gene bombardment using high velocity microprojectiles (Yang et al.,1990), and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu,1988). Some of these techniques may be successfully adapted for in vivoor ex vivo use.

Once the expression construct has been delivered into the cell thepolynucleotide encoding the gene of interest may be positioned andexpressed at different sites. In certain embodiments, the polynucleotideencoding the gene may be stably integrated into the genome of the cell.This integration may be in the cognate location and orientation viahomologous recombination (gene replacement) or it may be integrated in arandom, non-specific location (gene augmentation). In yet furtherembodiments, the polynucleotide may be stably maintained in the cell asa separate, episomal segment of DNA. Such nucleic acid segments or“episomes” encode sequences sufficient to permit maintenance andreplication independent of or in synchronization with the host cellcycle. How the expression construct is delivered to a cell and where inthe cell the polynucleotide remains is dependent on the type ofexpression construct employed.

In yet another embodiment of the invention, the expression construct maysimply consist of naked recombinant DNA or plasmids. Transfer of theconstruct may be performed by any of the methods mentioned above whichphysically or chemically permeabilize the cell membrane. This isparticularly applicable for transfer in vitro but it may be applied toin vivo use as well. Dubensky et al. (1984) successfully injectedpolyomavirus DNA in the form of calcium phosphate precipitates intoliver and spleen of adult and newborn mice demonstrating active viralreplication and acute infection. Benvenisty and Neshif (1986) alsodemonstrated that direct intraperitoneal injection of calciumphosphate-precipitated plasmids results in expression of the transfectedgenes. It is envisioned that DNA encoding a gene of interest may also betransferred in a similar manner in vivo and express the gene product.

In still another embodiment of the invention, transferring a naked DNAexpression construct into cells may involve particle bombardment. Thismethod depends on the ability to accelerate DNA-coated microprojectilesto a high velocity allowing them to pierce cell membranes and entercells without killing them (Klein et al., 1987). Several devices foraccelerating small particles have been developed. One such device relieson a high voltage discharge to generate an electrical current, which inturn provides the motive force (Yang et al., 1990). The microprojectilesused have consisted of biologically inert substances such as tungsten orgold beads.

Selected organs including the liver, skin, and muscle tissue of rats andmice have been bombarded in vivo (Yang et al., 1990; Zelenin et al.,1991). This may require surgical exposure of the tissue or cells, toeliminate any intervening tissue between the gun and the target organ,i.e., ex vivo treatment. Again, DNA encoding a particular gene may bedelivered via this method and still be incorporated by the presentinvention.

In a further embodiment of the invention, the expression construct maybe entrapped in a liposome. Liposomes are vesicular structurescharacterized by a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, 1991). Also contemplated are lipofectamine-DNA complexes.

Liposome-mediated nucleic acid delivery and expression of foreign DNA invitro has been very successful. Wong et al., (1980) demonstrated thefeasibility of liposome-mediated delivery and expression of foreign DNAin cultured chick embryo, HeLa and hepatoma cells. Nicolau et al.,(1987) accomplished successful liposome-mediated gene transfer in ratsafter intravenous injection.

In certain embodiments of the invention, the liposome may be complexedwith a hemagglutinating virus (HVJ). This has been shown to facilitatefusion with the cell membrane and promote cell entry ofliposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments,the liposome may be complexed or employed in conjunction with nuclearnon-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In yetfurther embodiments, the liposome may be complexed or employed inconjunction with both HVJ and HMG-1. Since these expression constructshave been successfully employed in transfer and expression ofpolynucleotides in vitro and in vivo, then they are applicable for thepresent invention. Where a bacterial promoter is employed in the DNAconstruct, it also will be desirable to include within the liposome anappropriate bacterial polymerase.

Other expression constructs which can be employed to deliver apolynucleotide encoding a particular gene into cells arereceptor-mediated delivery vehicles. These take advantage of theselective uptake of macromolecules by receptor-mediated endocytosis inalmost all eukaryotic cells. Because of the cell type-specificdistribution of various receptors, the delivery can be highly specific(Wu and Wu, 1993).

Receptor-mediated gene targeting vehicles generally consist of twocomponents: a cell receptor-specific ligand and a DNA-binding agent.Several ligands have been used for receptor-mediated gene transfer. Themost extensively characterized ligands are asialoorosomucoid (ASOR) (Wuand Wu, 1987) and transferrin (Wagner et al., 1990). Recently, asynthetic neoglycoprotein, which recognizes the same receptor as ASOR,has been used as a gene delivery vehicle (Ferkol et al., 1993; Peraleset al., 1994) and epidermal growth factor (EGF) has also been used todeliver genes to squamous carcinoma cells (Myers, EPO 0273085).

In other embodiments, the delivery vehicle may comprise a ligand and aliposome. For example, Nicolau et al. (1987) employed lactosyl-ceramide,a galactose-terminal asialganglioside, incorporated into liposomes andobserved an increase in the uptake of the insulin gene by hepatocytes.Thus, it is feasible that a polynucleotide encoding a particular genealso may be specifically delivered into a cell type such as lung,epithelial or tumor cells, by any number of receptor-ligand systems withor without liposomes. For example, epidermal growth factor (EGF) may beused as the receptor for mediated delivery of a polynucleotide encodinga gene in many tumor cells that exhibit upregulation of EGF receptor.Mannose can be used to target the mannose receptor on liver cells. Also,antibodies to CD5 (CLL), CD22 (lymphoma), CD25 (T-cell leukemia) and MAA(melanoma) can similarly be used as targeting moieties.

In certain embodiments, gene transfer may more easily be performed underex vivo conditions. Ex vivo gene therapy refers to the isolation ofcells from an animal, the delivery of a polynucleotide into the cells invitro, and then the return of the modified cells back into an animal.This may involve the surgical removal of tissue/organs from an animal orthe primary culture of cells and tissues.

6. Cellular Grafts

The present section provides a discussion of methods and compositionsfor grafting donor cells.

a. Preparation of Donor Cells

The donor cells need to be properly prepared for grafting, e.g., forinjection of genetically modified donor cells, fibroblasts obtained fromfor example, skin samples are placed in a suitable culture medium forgrowth and maintenance of the cells. Such a solution may contain fetalcalf serum (FCS) in which the cells are allowed to grow to confluence.

The cells are loosened from the culture substrate for example using abuffered solution containing 0.05% trypsin and placed in a bufferedsolution such as PBS supplemented with 5% serum to inactivate trypsin.The cells may be washed with PBS using centrifugation and thenresuspended in the complete PBS without trypsin and at a selecteddensity for injection. In addition to PBS, any osmotically balancedsolution which is physiologically compatible with the host subject maybe used to suspend and inject the donor cells into the host.

The long-term survival of implanted cells may depend on effects of theviral infection on the cells, on cellular damage produced by the cultureconditions, on the mechanics of cell implantation, or the establishmentof adequate vascularization, and on the immune response of the hostanimal to the foreign cells or to the introduced gene product. Themammalian brain has traditionally been considered to be animmunologically privileged organ, but recent work has shown conclusivelythat immune responses can be demonstrated to foreign antigens in the ratbrain. It is important to minimize the potential for rejection andgraft-versus-host reaction induced by the grafted cells by usingautologous cells wherever feasible, by the use of vectors that will notproduce changes in cell surface antigens other than those associatedwith the phenotypic correction and possibly by the introduction of thecells during a phase of immune tolerance of the host animal, as in fetallife.

The most effective mode and timing of grafting of the transgene donorcells of the invention to treat defects, disease or trauma in the CNS ofa patient will depend on the severity of the defect and on the severityand course of disease or injury to cells such as neurons in the CNS, thepatient's health and response to treatment and the judgment of thetreating health professional.

Of course, as in all other gene-transfer systems, the important issuesof appropriate or faithful gene expression must be resolved to ensurethat the level of gene expression is sufficient to achieve the desiredphenotypic effect and not so high as to be toxic to the cell.

A problem associated with the use of genetically engineered cells astransplants for gene therapy is that as cells become quiescent(non-dividing) the expression of genes, including transgenes, becomesdown-regulated (Palmer et al., 1991). Primary fibroblasts grafted intothe brain do not continue to divide when implanted unless they aretransformed and tumorigenic. They thus exist in a quiescent state in thebrain. It is thus useful to provide means for maintaining and/orincreasing expression of the transgene in the absence of cell divisionto promote long term stable expression of therapeutic genes used infibroblasts for gene therapy.

Expression of a gene is controlled at the transcription, translation orpost-translation levels. Transcription initiation is an early andcritical event in gene expression. This depends on the promoter andenhancer sequences and is influenced by specific cellular factors thatinteract with these sequences. The discussion regarding promoters andenhancers above is incorporated herein by reference.

The genetic correction of some, or many, CNS disorders may require theestablishment or re-establishment of faithful intercellular synapticconnections. Model systems to study these possibilities have not yetbeen developed and exploited because of the paucity of replicatingnon-transformed cell-culture systems and the refractoriness ofnon-replicating neuronal cells to retroviral infection. However, recentstudies, including those involving the immortalization of embryonichippocampal neuronal cells, suggest that replicating neuronal cellculture systems may soon become available for in vitro gene transfer andthen for in vivo implantation (Caettano and MacKay, 1990). Such neuronsmight be susceptible to efficient transduction by retroviral or otherviral vectors, and if they are also able to retain other neuronalcharacteristics, they may be able to establish synaptic connections withother cells after grafting into the brain. Alternatively, there arecells within the CNS that are late to develop, such as the ventral leafof the dentate gyrus of the hippocampus, or continue to divide throughadulthood, such as those in the olfactory mucosa and in the dentategyrus. Such cells may be suitable targets for retroviral infection.

The use of non-neuronal cells for grafting may preclude the developmentof specific neural connections to resident target cells of the host.Therefore, the phenotypic effects of fibroblast or other non-neuronaldonor cells or target cells in vivo would be through the diffusion of arequired gene product or metabolite, through gap junctions (“metabolicco-operation”) or through uptake by target cells of secreted donor cellgene products or metabolites.

Alternatively, neural bridges may be provided which facilitatereconnection between neurons in damaged CNS tissues. As noted above,grafted donor cells suspended in substrate material such as collagenmatrices can serve as neural bridges to facilitate axonal regenerationand reconnection of injured neurons, or may be used in conjunction withneural bridges formed from synthetic or biological materials, forexample homogenates of neurons or placenta, or neurite promotingextracellular matrices.

b. Grafting Donor Cells

The methods of the invention contemplate intracerebral grafting of donorcells containing a transgene insert to the region of the CNS havingsustained defect, disease or trauma. More specifically, the presentinvention contemplates grafting cells containing AADC/VMAT transgenes tosubjects exhibiting PD symptoms.

Neural transplantation or “grafting” involves transplantation of cellsinto the central nervous system or into the ventricular cavities orsubdurally onto the surface of a host brain. Conditions for successfultransplantation include: 1) viability of the implant; 2) retention ofthe graft at the site of transplantation; and 3) minimum amount ofpathological reaction at the site of transplantation.

Methods for transplanting various nerve tissues, for example embryonicbrain tissue, into host brains have been described in Neural Grafting inthe Mammalian CNS, Bjorklund and Stenevi, eds., (1985) Das, Ch. 3 pp.23-30; Freed, Ch. 4, pp. 31-40; Stenevi et al., Ch. 5, pp. 41-50;Brundin et al., Ch. 6, pp. 51-60; David et al., Ch. 7, pp. 61-70;Seiger, Ch. 8, pp. 71-77 (1985), incorporated by reference herein. Theseprocedures include intraparenchymal transplantation, i.e. within thehost brain (as compared to outside the brain or extraparenchymaltransplantation) achieved by injection or deposition of tissue withinthe host brain so as to be opposed to the brain parenchyma at the timeof transplantation (Das, 1985).

The two main procedures for intraparenchymal transplantation are: 1)injecting the donor cells within the host brain parenchyma or 2)preparing a cavity by surgical means to expose the host brain parenchymaand then depositing the graft into the cavity (Das, 1985). Both methodsprovide parenchymal apposition between the graft and host brain tissueat the time of grafting, and both facilitate anatomical integrationbetween the graft and host brain tissue. This is of importance if it isrequired that the graft become an integral part of the host brain and tosurvive for the life of the host.

Alternatively, the graft may be placed in a ventricle, e.g. a cerebralventricle or subdurally, i e. on the surface of the host brain where itis separated from the host brain parenchyma by the intervening pia materor arachnoid and pia mater. Grafting to the ventricle may beaccomplished by injection of the donor cells or by growing the cells ina substrate such as 3% collagen to form a plug of solid tissue which maythen be implanted into the ventricle to prevent dislocation of thegraft. For subdural grafting, the cells may be injected around thesurface of the brain after making a slit in the dura. Injections intoselected regions of the host brain may be made by drilling a hole andpiercing the dura to permit the needle of a microsyringe to be inserted.The microsyringe is preferably mounted in a stereotaxic frame and threedimensional stereotaxic coordinates are selected for placing the needleinto the desired location of the brain or spinal cord.

The donor cells may also be introduced into the putamen, nucleusbasalis, hippocampus cortex, striatum or caudate regions of the brain,as well as the spinal cord. Preferably, for passaged donor cells, cellsare passaged from approximately 2 to approximately 20 passages. Ofcourse it is understood that the cells may be passaged more than 20times and further it is specifically contemplated that the cells may bepassaged 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 ormore times.

For grafting, the cell suspension is drawn up into the syringe andadministered to anesthetized graft recipients. Multiple injections maybe made using this procedure. The age of the donor tissue, i.e. thedevelopmental stage may affect, the success of cell survival aftergrafting.

The cellular suspension procedure thus permits grafting of geneticallymodified donor cells to any predetermined site in the brain or spinalcord, is relatively non-traumatic, allows multiple graftingsimultaneously in several different sites or the same site using thesame cell suspension, and permits mixtures of cells from differentanatomical regions. Multiple grafts may consist of a mixture of celltypes, and/or a mixture of transgenes inserted into the cells.Preferably from approximately 10⁴ to approximately 10¹² cells areintroduced per graft. Thus it is envisioned that 10⁵, 10⁶, 10⁷, 10⁸ 10⁹10¹⁰ or 10¹¹ cells may be introduced per graft. Additionally it iscontemplated that more than one graft may be necessary, indeed 1, 2, 3,4, 5, 6, 7, 8, 9, 10 or more grafts may be performed over any givenperiod ranging from days to weeks to months to years.

For transplantation into cavities, which may be preferred for spinalcord grafting, tissue is removed from regions close to the externalsurface of the CNS to form a transplantation cavity, for example asdescribed by Stenevi et al., (1985), by removing bone overlying thebrain and stopping bleeding with a material such a gelfoam. Suction maybe used to create the cavity. The graft is then placed in the cavity.More than one transplant may be placed in the same cavity usinginjection of cells or solid tissue implants.

Grafting of donor cells into a traumatized brain will require differentprocedures, for example, the site of injury must be cleaned and bleedingstopped before attempting to graft. In addition, the donor cells shouldpossess sufficient growth potential to fill any lesion or cavity in thehost brain to prevent isolation of the graft in the pathologicalenvironment of the traumatized brain.

The present invention therefore provides methods for geneticallymodifying donor cells for grafting CNS to treat defective, diseasedand/or injured cells of the CNS.

The methods of the invention also contemplate the use of grafting oftransgenic donor cells in combination with other therapeutic proceduresto treat disease or trauma in the CNS. Thus, genetically modified donorcells of the invention may be co-grafted with other cells, bothgenetically modified and non-genetically modified cells which exertbeneficial effects on cells in the CNS, such as chromaffin cells fromthe adrenal gland, fetal brain tissue cells and placental cells. Thegenetically modified donor cells may thus be supported by the survivaland function of co-grafted, non-genetically modified cells.

Moreover, the genetically modified donor cells of the invention may beco-administered with therapeutic agents useful in treating defects,trauma or diseases of the CNS, such as growth factors, e.g nerve growthfactor; gangliosides; antibiotics, neurotransmitters, neurohormones,toxins, neurite promoting molecules; and antimetabolites and precursorsof these molecules such as the precursor of dopamine, L-DOPA.

c. Tissue Cell Culture

Primary mammalian cell cultures may be prepared in various ways. Inorder for the cells to be kept viable while in vitro and in contact withthe expression construct, it is necessary to ensure that the cellsmaintain contact with the correct ratio of oxygen and carbon dioxide andnutrients but are protected from microbial contamination. Cell culturetechniques are well documented and are disclosed herein by reference(Freshner, 1992).

One embodiment of the foregoing involves the use of gene transfer toimmortalize cells for the production of proteins. The gene for theprotein of interest may be transferred as described above intoappropriate host cells followed by culture of cells under theappropriate conditions. The gene for virtually any polypeptide may beemployed in this manner. The generation of recombinant expressionvectors, and the elements included therein, are discussed above.Alternatively, the protein to be produced may be an endogenous proteinnormally synthesized by the cell in question.

Examples of useful mammalian host cell lines are Vero and HeLa cells andcell lines of Chinese hamster ovary (CHO), W138, BHK, COS-7, 293, HepG2,NIH3T3, RIN and MDCK cells. In addition, a host cell strain may bechosen that modulates the expression of the inserted sequences, ormodifies and process the gene product in the manner desired. Suchmodifications (e.g., glycosylation) and processing (e.g., cleavage) ofprotein products may be important for the function of the protein.Different host cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins. Appropriatecell lines or host systems can be chosen to insure the correctmodification and processing of the foreign protein expressed.

A number of selection systems may be used including, but not limited to,HSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase andadenine phosphoribosyltransferase genes, in tk-, hgprt- or aprt-cells,respectively. Also, anti-metabolite resistance can be used as the basisof selection for dhfr, that confers resistance to; gpt, that confersresistance to mycophenolic acid; neo, that confers resistance to theaminoglycoside G418; and hygro, that confers resistance to hygromycin.

Animal cells can be propagated in vitro in two modes: as non-anchoragedependent cells growing in suspension throughout the bulk of the cultureor as anchorage-dependent cells requiring attachment to a solidsubstrate for their propagation (i.e., a monolayer type of cell growth).

Non-anchorage dependent or suspension cultures from continuousestablished cell lines are the most widely used means of large scaleproduction of cells and cell products. However, suspension culturedcells have limitations, such as tumorigenic potential and lower proteinproduction than adherent T-cells.

7. Methods for Screening Active Compounds

The present invention also contemplates the use of the geneticallyengineered cells of the present invention in the screening of compoundsfor activity in either stimulating dopamine production, uptake oractivity. These assays may make use of a variety of different formatsand may depend on the kind of “activity” for which the screen is beingconducted. Contemplated functional “read-outs” include binding to acompound, inhibition of binding to a substrate, ligand, receptor orother binding partner by a compound, enzyme activity assays in which thedecarboxylation or monoamine transport activity is monitored.Additionally, in vivo assays that monitor behavioral traits may be used,such traits include rotational behavior the swing behavior andperformance in radial maze tests (see e.g., Ungerstedt et al., 1973;McGurk et al., 1992; U.S. Pat. No. 5,135,956).

a. In Vitro Assays

In one embodiment, the invention will be applied to the screening ofcompounds that bind to the AADC or VMAT proteins or fragments thereof.The polypeptides or fragments may be either free in solution, fixed to asupport, or expressed in or on the surface of a cell. Either thepolypeptides or the compound may be labeled, thereby permitting adetermination of binding. A particularly preferred screening assay isthe biochemical screening assay described herein below in Example 4.

In another embodiment, the assay may measure the inhibition of bindingof either protein to a natural or artificial substrate or bindingpartner. Competitive binding assays can be performed in which one of theagents is labeled. Usually, the polypeptide will be the labeled species.One may measure the amount of free label versus bound label to determinebinding or inhibition of binding.

Another technique for high throughput screening of compounds isdescribed in WO 84/03564. Large numbers of small peptide test compoundsare synthesized on a solid substrate, such as plastic pins or some othersurface. The peptide test compounds are reacted with either protein andwashed. Bound polypeptide is detected by various methods.

Purified AADC or VMAT can be coated directly onto plates for use in theaforementioned drug screening techniques. However, non-neutralizingantibodies to the polypeptides can be used to immobilize thepolypeptides to a solid phase. Also, fusion proteins containing areactive region (preferably a terminal region) may be used to link theAADC or VMAT complex active region to a solid phase.

Various cell lines containing wild-type or natural or engineeredmutations in the AADC/VMAT combination can be used to study variousfunctional attributes of the proteins and how a candidate compoundaffects these attributes. Methods for engineering mutations aredescribed elsewhere in this document. In such assays, the compound wouldbe formulated appropriately, given its biochemical nature, and contactedwith a target cell. Depending on the assay, culture may be required. Thecell may then be examined using a number of different physiologicassays. Alternatively, molecular analysis may be performed in which thefunction of AADC/VMAT, or related pathways, may be explored. This mayinvolve assays such as those for protein expression, enzyme function,substrate utilization, cAMP levels, mRNA expression (includingdifferential display of whole cell or polyA RNA) and others.

Protein-protein interactions may also be studied by using biochemicaltechniques such as cross-linking, co-immunoprecipitation, andco-fractionation by chromatography, which are well known to thoseskilled in the art. The co-immunoprecipitation technique consists of (i)generating a cell lysate; (ii) adding an antibody to the cell lysate;(iii) precipitating and washing the antigen; and (iv) eluting andanalyzing the bound proteins (Phizicky and Fields, 1995). The antigenused to generate the antibody can be a purified protein, or a syntheticpeptide coupled to a carrier. Both monoclonal and polyclonal antibodiescan be utilized in co-immunoprecipitation, or alternatively, a proteincan be used which carries an epitope tag recognized by a commerciallyavailable antibody.

b. In Vivo Assays

Treatment of animals with the engineered cells of the present invention,or test compounds will involve the administration of the compound, in anappropriate form, to the animal. Administration will be by any route thecould be utilized for clinical or non-clinical purposes, including butnot limited to oral, nasal, buccal, rectal, vaginal or topical.Alternatively, administration may be by intratracheal instillation,bronchial instillation, intradermal, subcutaneous, intramuscular,intraperitoneal or intravenous injection. Specifically contemplated aresystemic intravenous injection, regional administration via blood orlymph supply and intracerebral injection.

Determining the effectiveness of a therapy in vivo may involve a varietyof different criteria. Such criteria include, but are not limited to,survival, reduction of neurodegeneration, arrest or slowing of PDrelated tremor, elimination or reduction of other PD related symptoms,increased activity level, improvement in motor skills, improvement inalertness, improvement in sleeping habits, increased energy level,improvement in overall well-being, improvement in immune effectorfunction and improved food intake.

Particularly preferred in vivo assays for determining the effectivenessof the present invention are the behavioral tests described herein belowin Example 5. For example, monitoring improvements in deficits inforepaw adjusting steps in models will be a useful tool forinvestigating the efficacy of therapeutics (Chang et al., 1999). Otherbehavioral tests well known to those of skill in the art also arecontemplated.

8. Methods for Treating Parkinson's Disease

The present invention also involves, in another embodiment, theprevention or treatment of PD in a subject. By treatment, it is intendedthat the present invention will provide some alleviation of the symptomsof PD. Thus, it is not a requirement that all of the symptoms of PD beremoved. Since the cells generated by the present invention have acapacity to produce and sequester dopamine, it is contemplated thatgrafting such cells into a subject presenting PD will ameliorate some orall of the deleterious effects of PD. Alternatively, the cells of thepresent invention also may be used to identify additional therapeuticagents that will act to enhance dopamine production and/or release.

The present invention includes, in specific embodiments, methods for thetreatment of PD through the delivery of cells expressing functional AADCand VMAT transgenes. These cells will be used for the production andsubsequent storage and secretion of dopamine. The L-DOPA that isconverted to dopamine may be endogenous to the organism or may beprovided from an exogenous source in combination with the geneticallymodified cells of the present invention.

In many contexts, it is not necessary that all of the symptoms of PD bealleviated. Rather, to accomplish a meaningful treatment, all that isrequired is that the effects of this neurodegenerative disease be slowedto some degree. It may be either that the cells of the present inventionincrease the individuals ability to synthesize dopamine, store dopamineor merely provide enough dopamine to alleviate the symptoms of PD.Clinical aspects of PD are well known and defined in the art and includetremor (or trembling) of the arms and legs, stiffness and rigidity, lossof postural reflexes, and slowness of movement as well as other symptomsdescribed above.

a. Genetic Based Therapies

One of the therapeutic embodiments contemplated by the present inventionis the intervention, at the molecular level, in events involved in PD.Specifically, the present inventors intend to provide, to a cell or ananimal patient, an composition comprising a host cells that comprises anexpression construct capable of producing AADC and VMAT. Additionally,it is contemplated that a nucleic acid encoding TH and GTPcyclohydrolase I also may be included in the expression construct.Expression of these molecules will provide healthy cells with increasedproduction and/or secretion of the neurotransmitter dopamine. Thelengthy discussion of polynucleotides employed herein is incorporatedinto this section by reference. Particularly preferred polynucleotidesare contained in viral vectors such as adenovirus, adeno-associatedvirus, herpesvirus, vaccinia virus, parvovirus and retrovirus.

Those of skill in the art are well aware of how to apply gene deliveryto in vivo and ex vivo situations. For viral vectors, one generally willprepare a viral vector stock. Depending on the kind of virus and thetiter attainable, one will deliver 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸,1×10⁹, 1×10¹⁰, 1×10¹¹ or 1×10¹² infectious particles to the cell.Similar figures may be extrapolated for other non-viral formulations bycomparing relative uptake efficiencies.

b. Agents that Alleviate PD

Agents or factors suitable for use in a combined therapy with cell-basedtherapies of the present invention are any chemical compound ortreatment method that alleviates the deleterious effects of PD. Suchagents and factors include dopamine, L-DOPA, remacemide, benzamidecompounds, levodopa alone or in combination with a DOPA decarboxylaseinhibitor such as carbidopa or benserazide. These agents for PD therapyare extensively described in the literature e.g., U.S. Pat. No.5,017,607; U.S. Pat. No. 4,826,875; U.S. Pat. No. 4,873,263; U.S. Pat.No. 4,663,349; U.S. Pat. No. 4,771,073; U.S. Pat. No. 5,607,969; U.S.Pat. Nos. 5,817,690, 5,817,699, 5,807,871, 5,756,550; 5,756,548.Additional agents known to be useful in the treatment of PD include2-arylamidothiazole derivatives (U.S. Pat. No. 5,712,270); modulators ofacetylcholine receptors (U.S. Pat. No. 5,703,100); peptide mimeticdopamine prodrugs (U.S. Pat. No. 5,686,423); ATP-sensitive potassiumchannel blocker (U.S. Pat. No. 5,677,344); riluzole (U.S. Pat. No.5,674,885); carbamazepine and oxcarbazepine (U.S. Pat. No. 5,658,900);remacemide (U.S. Pat. No. 5,650,443); xanthine derivatives (U.S. Pat.No. 5,587,378); N-propargyl-aminoindan compounds (U.S. Pat. No.5,576,353); therapeutic purine agents (U.S. Pat. No. 5,565,460);nicotinamide adenine dinucleotide agents (U.S. Pat. No. 4,970,200);melanin and melanin-related compounds (U.S. Pat. No. 5,210,076); phyticacid (U.S. Pat. No. 5,206,226); pentanedione derivatives (U.S. Pat. No.5,112,861); D-DOPA (U.S. Pat. No. 4,863,962); Pterin derivatives (U.S.Pat. No. 4,758,571). Each of these patents is specifically incorporatedherein by reference as disclosing compositions that may be used incombination with the therapeutic compositions described in the presentinvention.

The skilled artisan is directed to “Remington's Pharmaceutical Sciences”15th Edition, chapter 33, in particular pages 624-652. Some variation indosage will necessarily occur depending on the condition of the subjectbeing treated. The person responsible for administration will, in anyevent, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biologics standards.

The inventors propose that the delivery of AADC/VMAT polynucleotides topatients will be an efficient method for delivering an effective therapyto counteract the effects of the nuerodegeneration seen in PD.Similarly, the drugs discussed herein above may be directed to aparticular, affected region of the subject's body or be administeredsystemically.

c. Combined Therapy with Traditional PD Therapies

One goal of current research is to find ways to improve the efficacy oftraditional PD therapy. One way is by combining such traditionaltherapies with gene therapy by grafting cells of the present inventioninto an animal exhibiting PD symptoms. In the context of the presentinvention, it is contemplated that the cell-based therapies of thepresent invention could be used in conjunction with any of the PDtherapies described above.

To achieve a therapeutic outcome and alleviate some or all of thesymptoms of PD using the methods and compositions of the presentinvention, one would generally graft the genetically modified donor cellinto the brain area of the PD subject and contact said cell or subjectat least one other PD therapeutic agent. These compositions would beprovided in a combined amount effective to induce an increase in theproduction, storage and/or secretion of dopamine. This process mayinvolve grafting the genetically cells and providing the agent(s) orfactor(s) at the same time. This may be achieved by contacting the PDindividual with a single composition or pharmacological formulation thatincludes both the cellular graft and the additional therapeuticagent(s), or by contacting the PD subject with two distinct compositionsor formulations, at the same time, wherein one composition includes thecellular composition and the other includes the agent.

Alternatively, the cell-based treatments may precede or follow the othertherapeutic agent treatment by intervals ranging from minutes to weeks.In embodiments where the other agent and the cell-based therapy areapplied separately to the PD subject, one would generally ensure that asignificant period of time did not expire between the time of eachdelivery, such that the agent and graft would still be able to exert anadvantageously combined effect on the subject. In such instances, it iscontemplated that one would contact the subject with both modalitieswithin about 12-24 hours of each other and, more preferably, withinabout 6-12 hours of each other, with a delay time of only about 12 hoursbeing most preferred. In some situations, it may be desirable to extendthe time period for treatment significantly, however, where several days(2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapsebetween the respective administrations.

It is also conceivable that more than one administration of either thecellular graft, or the other agent will be desired. Various combinationsmay be employed, where graft is “A” and the other therapeutic agent is“B”, as exemplified below:

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/BA/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B B/A/A/A A/B/A/A A/A/B/AA/B/B/B B/A/B/B B/B/A/B

Other combinations are contemplated. Again, to achieve a therapeuticoutcome, both agents are delivered to a cell in a combined amounteffective to increase or enhance the production and/or storage ofdopamine.

d. Formulations and Routes for Administration

Where clinical applications are contemplated, it will be necessary toprepare pharmaceutical compositions—expression vectors, cellular grafts,proteins, antibodies and drugs—in a form appropriate for the intendedapplication. Generally, this will entail preparing compositions that areessentially free of pyrogens, as well as other impurities that could beharmful to humans or animals.

One will generally desire to employ appropriate salts and buffers torender delivery vectors stable and allow for uptake by target cells.Buffers also will be employed when recombinant cells are introduced intoa patient. Aqueous compositions of the present invention comprise aneffective amount of the vector to cells, dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium. Such compositionsalso are referred to as inocula. The phrase “pharmaceutically orpharmacologically acceptable” refer to molecular entities andcompositions that do not produce adverse, allergic, or other untowardreactions when administered to an animal or a human. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents and the like. The use of suchmedia and agents for pharmaceutically active substances is well know inthe art. Except insofar as any conventional media or agent isincompatible with the vectors or cells of the present invention, its usein therapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions.

In addition to the cellular grafts of the present invention, additionalactive compositions described herein above present invention may includeclassic pharmaceutical preparations. Administration of thesecompositions according to the present invention will be via any commonroute so long as the target tissue is available via that route. Thisincludes oral, nasal, buccal, rectal, vaginal or topical. Alternatively,administration may be by orthotopic, intradermal, subcutaneous,intramuscular, intraperitoneal or intravenous injection. Suchcompositions would normally be administered as pharmaceuticallyacceptable compositions, described above.

The active compounds also may be administered parenterally orintraperitoneally. Solutions of the active compounds as free base orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial an antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

9. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Materials and Methods

Cell Culture

Fibroblast cells (2.5×10⁵) were plated in 6 well plates in DMEM mediawith 10% fetal calf serum with appropriate selection markers asdescribed in (Kang et al., 1993, Bencsics et al., 1996, Wachtel et al.,1997). Catecholamine measurements were done in DMEM media (1 ml in eachwell) except for the experiments on Ca⁻⁺ effect which were done inphysiological incubation medium consisting of 135 mM NaCl, 3 mM KCl, 2mM CaCl₂, 2 mM MgCl₂, 10 mM glucose, 200 μM ascorbic acid, and 10 mMHEPES, pH 7.3.

Generate of PFVMAA Cells

A full-length bovine AADC cDNA was cloned into a retroviral vector(LDcSHL; with selection marker hygromycin-B-phosphotransferase under thecontrol of an internal simian virus 40 early promoter) as described(Kang et al., 1993). The 1.6 kilobase fragment containing the fullcoding lesion and the part of the 3′ untranslated lesion of rat VMAT-2cDNA (a gift from Robert Edwards, University of California, SanFrancisco, Calif.) was removed by EcoRI, blunted and ligated into LINX(a gift from Fred H. Gage, Salk Biological Institute, La Jolla, Calif.,as described by (Hoshimaru et al., 1996) downstream from the P_(hCMV*−1)promoter. Both were used to generate amphotropic retroviral producerlines from PA317 cells (PAAADC and PAVMAT). Isolation and culture ofprimary fibroblast from inbred Fischer 344 rats were describedpreviously (Kang et al., 1993, Bencsics et al., 1996, Wachtel et al.,1997). Primary fibroblasts (PF) were infected with either PAAADC orPAVMAT, and selected with hygromycin (150 μg/ml) or G418 (400 μg/ml) toestablish PFAADC or PFVMAT cells, respectively. PFAADC was infected withPAVMAT and underwent additional selection in media containing G418 andhygromycin to generate bulk population of primary fibroblast cellsexpressing both AADC and VMAT-2 (PFVMAA).

Immunostaining Studies

Control and transduced fibroblasts were grown on chamber slide(Lab-Tek), fixed with 4% phosphate-buffered parafornaldehyde, andperrneabilized with 0.2% Triton X-100. Cells were immunostained with arabbit polyclonal antibody against a synthetic peptide at the C terminusof VMAT-2 (Phoenix Pharmaceuticals, Inc., Calif.) at a dilution of1:5,000 or a polyclonal antibody against bovine AADC (CA-201 bDCrab,Protos Biotech Corp., NY) at a dilution of 1:1,000, and a biotinylatedgoat anti-rabbit IgG secondary antibody. The signal was amplified byavidin and biotinylated horseradish peroxidase using the Elite ABCVectastatin Kit (Vector Labs, CA.). VMAT-2 and AADC immunoreactivitywere visualized using 3,3′-diaminobenzidine tetrachloride dehydrate(Aldrich Chemical Co., WI) as a chromogen and enhanced by the additionof cobalt chloride/nickel ammonium.

Biochemical Assays

AADC activity was assayed with modification of a CO₂ trapping asdescribed, but without using radioactivity (Kang et al., 1993).Supernatant of homogenized cells was added to reaction solutioncontaining 30 mM potassium phosphate buffer, 0.3 mM EDTA, 20 μMpyridoxal 5′-phosphate, and 200 μM L-DOPA. Dopamine levels afterreaction were measured by reverse-phase HPLC using a Velosep RP-18column (100×3.2 mm; Applied Biosystems, Inc., CA) and an ESA CoulochemII electrochemical detector equipped with a 5014 analytical cell. Fortransporter assay, cells were washed and homogenized as described(Gasnier et al, 1994; Merickel et al., 1995). Cell debris was removed bycentrifugation in a microcentrifuge at 4000×g for 5 min. A Bradfordassay (Bio-rad) was performed to measure the protein concentration, andsupernatant was diluted in SH (sucrose HEPES) buffer to a finalconcentration of 10 mg/ml. From each transfection, two aliquots weremade and frozen at −80° C. An aliquot of frozen membranes was thawed and20 μl added to 200 μl of SH buffer containing 4 mM KCl, 2.5 mM MgSO₄, 2mM ATP (potassium salt), and 50 nM [³H]serotonin (DuPont NEN) at 29° C.for 5 minutes. The termination of reaction, filtration and radioactivitymeasurement was performed by the methods as previously described(Gasnier et al, 1994; Merickel et al., 1995). Experiments were performedin triplicates, and background uptake at 0° C. at 0 min was subtracted.

In Vivo Studies

The in vivo protocols were approved by the Institutional Animal Care andUse Committee of the University of Chicago. For the dopamine depletion,female Fischer 344 rats (150-200 g) were anesthetized with a mixture ofketamine (75 mg/kg), acepromazine (0.75 mg/kg) and xylazine (3.8 mg/kg).As previously described (Wachtel et al., 1997) for the medial forebrainbundle (MFB) lesion, 8 μg (free base weight) of 6-hydroxydopamine wasinfused unilaterally in 2 μl at a rate of 0.5 μl/min at the followingcoordinate: AP 4.4 mm, ML 1.2mm relative to bregma, and −7.5 mm from thedura. Animals with near complete lesions were used for furtherexperiments: rats with more than 400 rotations per hour afterD-amphetamine (5 mg/kg, i.p.) administration was used for themicrodialysis study and rats with less than 3 forepaw adjusting stepsover 12 seconds along 90 cm distance was used for behavioral study. PF,PFAADC, and PFVMAA cell were washed, trypsinized, and suspended inDulbecco's phosphate-buffered saline. Two microliters of the cellsuspension (75,000 cells/μl) was infused at each of four ventral sites(AP 1.2 and −0.3, ML 2.3 and 3.0, DV −4.0) and 1 μl at another fourdorsal sites within the same needle tracts (DV −3.5) for a total of900,000 cells per animal.

Microdialysis

Microdialysis probes were implanted at the center of 4 tracks of grafts(AP 0.45, ML 2.65, DV 5.2) under anesthesia and the microdialysis probeswere of vertical concentric design as previously described (Wachtel etal., 1997). Before implantation, the microdialysis probes (2 mm activearea) were calibrated in vitro for relative recovery to assureconsistency, but the data were not corrected for recovery. Artificialcerebrospinal fluid (147 mM NaCl, 2.5 mM KCl, 1.3 mM CaCl₂, and 0.9 mMMgCl₂, pH≈7.4) was infused continuously through the probe at a rate of1.5 μl/min. The day after probe placement, dialysates were collected at20-min intervals for 340 min after L-DOPA administration in awake,freely moving animals. Twenty microliters of each dialysate sample wasanalyzed by HPLC for L-DOPA, dopamine, and DOPAC concentrations.

Behavioral Testing

For the behavioral test, we used the coordinates and amount of infusedcells optimized to influence forepaw adjusting step with minimalnonspecific effect on the behavior from the graft mass based on ourlocalization of areas in the striatum crucial for the behavior (Olssonet al., 1995). 0.67 μl of the cell suspension (75,000 cells/μl) wereinfused at each of two VL sites (AP 1.1, ML 3.2, DV 5.7 and 5.0), threeVL lateral sites (AP 1.1, ML 4.2, DV 6.0, 5.3, and 4.6), and three VLanterior medial sites (AP 2.2, ML 2.2, DV 5.7, 5.0, and 4.3) for a totalof 400,000 cells per animal. The cell suspensions were injected at therate of 0.5 μl/min using a 10 μl syringe and an infusion pump. Graftingwas only done with cells below passage 15. The experimenter holds therat's hindlimbs and one forepaw, so that the animal must bear its weightsolely with its opposing forelimb on the treadmill belt. The number ofcatch-up steps made while the belt moved 90 cm/12 seconds were countedmanually as described (Olsson et al., 1995; Chang et al., 1999). Thestepping numbers over five intervals were then averaged for eachforepaw. Only rats with complete lesions (from 0 to 3 steps/interval inthe contralateral forepaw) were used for experiments. Rats wereadministered L-DOPA (6 mg/kg) and benserazide (50 mg/kg), and checkedfor stepping responses at one hour intervals for 6 hours.

Immunohistology

One day after microdialysis and behavioral experiments, rats wereanesthetized and transcardially perfused with 125 ml of normal salinefollowed by 250 ml of ice-cold 4% paraformaldehyde. Brains were removed,postfixed for overnight, and transferred to 30% sucrose untilequilibrated. Forty-micrometer sections were cut and stained with Nisslor immunostained with AADC and VMAT-2 antibodies herein.

Example 2 Transgene Expression in Fibroblasts

Primary skin fibroblast cells (PF) from Fischer 344 rats weregenetically modified to express AADC (PFAADC) or both VMAT-2 and AADC(PFVMAA) (see Example 1). Expressions of functional transgenes wereconfirmed by immunohistochemistry and activity assays (FIG. 1A-FIG. 1Fand Table 2). The fibroblast cells were incubated with 1 μM L-DOPA, andthe dopamine levels both in the cells and media were measured.Previously it has been shown that the control fibroblasts do not produceany detectable dopamine from L-DOPA (Kang et al., 1993). Consistent withprevious data, the intracellular storage of dopamine in PFAADC cellswere negligible (FIG. 2A) and most of dopamine was released into theextracellular space, increasing with time (FIG. 2B). Extracellulardopamine level in the media of PFVMAA cells also continued to increasewith time and was significantly higher than that of PFAADC cells (FIG.2B). Intracellular dopamine levels in PFVMAA reached a plateau after 2hours, indicating saturation of the storage capacity for dopamine (FIG.2A). When fibroblast cells with only VMAT expression were incubated with1 μM dopamine in the media for an hour, intracellular dopamine levelswere 22.5±2.5 pmoles/10⁶ cells (mean±SEM, n=3) compared to 122.1±6.8 atthe same one hour time point in PFVMAA cells (FIG. 2A). Given the factthat both PFVMAA and PFVMAT cells have similar storage capacities(transporter activity of PFVMAT cells; pmoles 5-HT/mg/5min), thisdifference in the dopamine levels indicates that using L-DOPA as aprecursor along with AADC for intracellular conversion into dopamineresults in much higher levels of dopamine storage than using dopamineitself.

Example 3 Increased Dopamine Production is Due to VMAT-2 Expression

To further demonstrate that the increased dopamine production andstorage were due to solely VMAT-2 expression, cells were incubated withL-DOPA (1 μM) and a VMAT inhibitor, reserpine (3 μM) for 2 hours anddopamine levels were measured. Reserpine depleted intracellular dopaminefrom PFVMAA completely and also decreased extracellular dopamine levelof PFVMAA to the levels comparable to that of PFAADC cells (FIG. 2C).Total dopamine production in PFVMAA cells was higher than that in thesame cells treated with reserpine or in PFAADC cells (FIG. 2C) despitecomparable AADC activities, suggesting that sequestration of thedopamine away from the site of synthesis facilitates total dopamineproduction. DOPAC was not detected in PFVMAA cells and reserpinetreatment of PFVMAA cells increased DOPAC levels to that of PFAADC (FIG.2D). This is consistent with metabolism of cytoplasmic dopamine bymonoamine oxidase (MAO) in PFAADC cells or PFVMAA cells with reserpine.On the other hand, dopamine is protected from the metabolism because itis sequestrated in the vesicles of PFVMAA cells. The lack of metabolismof dopamine contributes to higher total dopamine in PFVMAA, but does notaccount for the entire difference from other situations noted above. Insummary, VMAT-2 not only increases the intracellular storage capacityand reduce metabolism of dopamine, but also increase overall productionof dopamine in fibroblasts genetically modified with AADC (see Table 2).

TABLE 2 Activities of recombinant enzymes in genetically modifiedfibroblasts (n = 3) PF PFAADC PFVMAA AADC activity 0 323.98 300.51(pmole dopamine/mg/min) (±9.06) (±26.74) VMAT activity 0 0 48.6(±12.3)(pmole 5-HT/mg/5 min)

Example 4 Dopamine Release from PFVMAA Cells

Dopamine stored in PFVMAA cells was released spontaneously and graduallyover a few hours in the absence of continuing supply of the precursorL-DOPA (FIG. 3A). Time course of the dopamine release was not performedin PFAADC cells because there was no detectable intracellular dopaminein PFAADC cells after incubation with L-DOPA (FIG. 2A and Kang et al.,1993). To investigate the mechanism of dopamine release in the PFVMAAcells, intracellular calcium was manipulated by using calcium-free mediaand calcium ionophore. There was significant decrease in dopaminerelease by calcium depletion and a trend for increased release ofdopamine by calcium ionophore (FIG. 3B), suggesting that some of thevesicular release is calcium-dependent. However, the major portion ofrelease was calcium-independent and incubation with high potassium media(40 mM KCl) for 3 minutes did not increase dopamine release.Calcium-independent release of other classic neurotransmitters andpeptides from genetically modified fibroblast cells without addedstorage capacity has been noted previously (Ruppert et al., 1993. Misawaet al., 1994). The majority of dopamine release from PFVMAA is mostlikely due to constitutive exocytosis of vesicles. VMAT-2 has been foundin tubulovesicular organelles of cell bodies and dendrites ofdopaminergic neurons (Hattori et al., 1979; Mercer et al., 1979;Nirenberg et al., 1996). Somatodendritic release of dopamine fromdopaminergic neurons may occur from this pool of dopamine, in a similarmanner as dopamine release from PFVMAA cells. Likewise, Ca⁺⁺-independentconstitutive release of catecholamine has been noted in PC-12 cells andchromaffin cells (von Grafenstein et al., 1992; Sulzer et al., 1996).

Example 5 Biochemical Effect of PFVMAA Cell Graft in Parkinsonian Rats

The inventors tested whether PFVMAA cells will prolong the duration ofthe effect of systemically administered L-DOPA in vivo in terms ofdopamine release and behavioral restoration. Rats with unilateralnear-complete lesions of nigrostriatal system were produced by injecting6-hydroxydopamine in the medial forebrain bundle. Grafts were placed inthe central area of the striatum along four tracks. Three days later, amicrodialysis probe was placed at the center of 4 tracks of grafts andthe next day, catecholamine levels were monitored after intraperitonealadministration of L-DOPA and benserazide. AADC gene transfer achieved bythe PFAADC cells was not sufficient to increase the dopamine levelsproduced by the exogenous L-DOPA significantly over the level producedby endogenous AADC in the denervated striatum with the control grafts(FIG. 4A-FIG. 4C). This indicates that the relative contribution of AADCactivity provided by the PFAADC grafts is not significant compared tothe endogenous capacity to decarboxylate L-DOPA. Significant increasesin the level and duration of dopamine elevation were noted in PFVMAAgrafted striatum compared to control and PFAADC groups (FIG. 4A-FIG.4C). To exclude the possibility that the longer duration of dopamineelevation is simply due to a higher peak level, the duration of dopamineelevation in PFAADC group treated was compared with higher dose ofL-DOPA (25 mg/kg) that attained the same peak dopamine levels as thePFVMAA group given 6 mg/kg of L-DOPA. The duration of dopamine elevationwas significantly longer in PFVMAA group than PFAADC group with higherprecursor administration (FIG. 4B). This data is consistent withprolonged dopamine release by PFVMAA cells in addition to increasedtotal dopamine level. Also, it was observed that DOPAC levels weresignificantly lower in the PFVMAA grafted striatum than all the othergroups (FIG. 4C), which is consistent with the fact that VMAT protectsdopamine from MAO-mediated metabolism by sequestering it fromcytoplasmic space into vesicles.

Example 6 Behavioral Effect of PFVMAA Cell Graft in Parkinsonian Rats

To determine whether such prolongation of biochemical effect fromgrafting PFVMAA cells would also increase the duration of the effect onakinesia of the Parkinsonian rats, the forepaw adjusting steps wereutilized as a non-drug-induced behavioral paradigm that reflectsdopamine depletion and/or restoration more faithfully than rotationalbehaviors and reflects akinesia rather than compensatory supersensitivechanges (Olsson et al., 1995; Chang et al., 1999). PF, PFAADC, andPFVMAA cells were grafted into denervated striatum and forepaw adjustingsteps were monitored for six hours after administration of L-DOPA (6mg/kg) and benserazide (50 mg/kg) on the seventh day after grafting.Compared to the baseline level before L-DOPA injection, forepawadjusting steps were significantly elevated for 4 hours in PFVMAAgrafted rats, for 2 hours in both PFAADC and PF groups. Again PFAADC didnot show significant difference from the control group (FIG. 4D). Thegenetically modified grafts survived well as noted previously (Bencsicset al., 1996; Wachtel et al., 1997) and expression of transgenes werealso confirmed by immunostaining for AADC and VMAT-2 (FIG. 5A-FIG.5H).

Example 7 Discussion

Addition of storage capacity by VMAT gene transduction significantlyincreased the peak dopamine levels and duration of its release both invitro and in vivo from the same dose of L-DOPA. This translated intoprolongation of the duration of improvement in akinesia. These findingsunderline the importance of dopamine storage capacity in the efficacy ofL-DOPA therapy. There are clinical examples that underscore theimportance of subsequent processing steps of L-DOPA, namely L-DOPAdecarboxylation and dopamine storage capacity. DOPA-responsive dystoniais associated with mutations in GTP cyclohydrolase 1 gene that leads toabsence of cofactor, tetrahydrobiopterin and consequent lack of dopamineproduction (Ichinose et al., 1994; Furukawa et al., 1996). Dramatic andsmooth response to L-DOPA in these patients is most likely due to theirintact L-DOPA decarboxylation and dopamine storage capacity (Nygaard etal., 1992; Snow et al., 1993; Turjanski, et al., 1993). The major effectof fetal transplantation in PD has been in enhancing patients' responseto L-DOPA, rather than alleviating the need for the drug and is againlikely to be due to added capacity to decarboxylate L-DOPA and store theformed dopamine which has been demonstrated by fluoro-DOPA PET scans(Lindvall et al, 1994; Sawle et al, 1992).

In addition to the prolongation of the effect of L-DOPA to alleviate theshort duration response, use of these grafts along with L-DOPA therapyin early to moderate stages of the disease may prevent the developmentof fluctuations as the PD advances. By allowing a lower dose of L-DOPAto be used in conjunction with the grafts to achieve the same level ofdopamine as a higher dose of L-DOPA, it may also reduce side effectsthat occur due to the diffusion of L-DOPA to other parts of the brain,notably limbic system, such as hallucinations and confusions. Suchdouble gene transduction could be applied to other donor cell types fortransplantation including neuronal stem cells as well as to direct invivo transfer methods using viral vectors such as herpes virus,adenovirus, adeno-associated virus, lentivirus. In addition, genes thatare essential for L-DOPA production such as tyrosine hydroxylase and GTPcyclohydrolase 1 (Bencsics et al., 1996) can be combined with AADC andVMAT-2 for production and delivery of dopamine by gene therapy. However,regulation of the optimal levels of dopamine requires additionalmeasures such as use of regulatable promoters. On the other hand, thecombination of precursor delivery and AADC/VMAT-2 double genetransduction employed here allows one of skill in the art to regulatethe final dopamine delivery by varying the amount of L-DOPA given. Inconclusion, key processing steps of L-DOPA outlined herein may lead tosuccessful amelioration of symptoms of PD by improving the major problemof the current mainstay of therapy.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

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What is claimed is:
 1. A method of increasing the efficiency of L-DOPAconversion into dopamine in mammal comprising: (a) obtaining cells fromsaid mammal; (b) transforming said cells in vitro with a firstpolynucleotide encoding L-amino acid decarboxylase (AADC) and a secondpolynucleotide encoding VMAT vesicular monoamine transporter (VMAT)under conditions suitable for the expression of AADC and VMAT, whereinsaid polynucleotides each are under transcriptional control of apromoter; (c) implanting transformed cells into said mammal; and (d)providing L-DOPA to said mammal, whereby AADC converts L-DOPA in vivo todopamine and VMAT sequesters said dopamine in endosomes of said cells,which sequestered dopamine releases over a longer duration of time thanfrom cells without storage of L-DOPA.
 2. The method of claim 1, whereinsaid first and second polynucleotides are covalently attached.
 3. Themethod of claim 2, wherein said first and second polynucleotides arepart of a viral vector.
 4. The method of claim 3, wherein said viralvector is selected from the group consisting of retrovirus, adenovirus,herpes virus, adeno-associated virus and lentivirus.
 5. The method ofclaim 2, wherein said first and second polynucleotides are under thecontrol of different promoters.
 6. The method of claim 2, wherein saidfirst and second polynucleotides are under the control of the samepromoter and are separated by an internal ribosome entry site.
 7. Themethod of claim 6, wherein said promoter is a tissue specific promoter.8. The method of claim 6, wherein said promoter is an induciblepromoter.
 9. The method of claim 6, wherein said promoter is aconstitutive promoter.
 10. The method of claim 6, wherein the promoteris selected from the group consisting of CMV IE, SV40 IE, β-actin,EF1-α, a TH promoter, AADC non-neuronal promoter; an AADC promoter, aVMAT2 promoter, a GTP cylohydrolase I promoter and a dopaine transporterpromoter.
 11. The method of claim 2, wherein said first and secondpolynucleotides each are covalently linked to a polyadenylation signal.12. The method of claim 1, wherein said cells are fibroblast cells. 13.The method of claim 12, wherein said cells are selected from the groupconsisting of fetal brain cell, an astrocyte, a neuronal cell, amyoblast, and a bone marrow stromal cell.
 14. The method of claim 1,further comprising transforming said cells with a polynucleotideencoding tyrosine hydroxylase (TH) wherein said TH encodingpolynucleotide is under the transcriptional control of a promoter. 15.The method of claim 14, further comprising transforming said cells witha polynucleotide encoding GTP cyclohydrolase I (GTPCH), wherein saidGTPCH encoding polynucleotide is under the transcriptional control of apromoter.
 16. The method of claim 1, wherein said L-DOPA is administeredby a route selected from the group consisting of orally, sublingually,subcutaneously, intravenously and by duodenal infusion.
 17. The methodof claim 16, wherein said L-DOPA is administered in a dose of betweenabout 50 to about 2500 mg of L-DOPA per day.
 18. The method of claim 17,further comprising administering carbidopa at a dose of between about 20to about 300 mg carbidopa per day.
 19. The method of claim 1, whereinsaid transformed cells are administered via stereotactic surgery to thebrain.
 20. A mammalian cell transformed with a first polynucleotideencoding aromatic L-amino acid decarboxylase (AADC) and a secondpolynucleotide encoding vesicular monoamine transporter (VMAT), whereinthe transformed cell produces dopamine when contacted with L-DOPA, whichsequestered dopaine releases over a longer duration of time than fromcells without storage of L-DOPA.
 21. The mammalian cell of claim 20 is afibroblast.
 22. The mammalian cell of claim 20, wherein the first andsecond polynucleotide are part of the same viral vector.